Biological applications of alkaloids derived from the tunicate Eudistoma sp.

ABSTRACT

Biological applications of synthetic and natural alkaloids derived from the tunicate Eudistoma sp. are disclosed. A method regulating cell growth includes contacting one or more cells with an effective concentration of a compound for regulating cell growth. These compounds include: Segoline A, Segoline B, Isosegoline A, Norosegoline, Debromoshermilamine, Eilatin, 4-methylpyrido[2,3,4-kl]acridine, pyrido[2,3,4-kl]acridine, 1-acetyl-2,6-dimethylpyrido[2,3,4-kl]acridine, and derivatives and combinations of these compounds. An effective concentration range for using these compounds can range from approximately 0.1 μM to 100 μM. The effective concentration range for Eilatin, the most potent of these compounds is from 0.01 μM to 0.99 μM, and the effective concentration range for the other compounds of the present invention is from about 1.0 μM to 100 μM. The method has been shown to suppress growth of tumor cells, to induce differentiation of the tumor cells, and induce reverse transformation of the tumor cells. In transformed cells, the method induces reverse transformation. The method also inhibits the proliferation of cells. The examples show that the method of the present invention affects cyclic AMP mediated biological processes. At the effective concentrations this method affects the cyclic AMP mediated biological processes of cells to achieve the results described above.

This is a continuation-in-part of application Ser. No. 07/924,194 filedon Aug. 3, 1992, now U.S. Pat. No. 5,278,168.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to biological applications of alkaloids derivedfrom the tunicate Eudistoma sp. and related compounds, to thepreparation of synthetic pyridoacridines, and particularly to thesynthesis of Eilatin.

2. Background of the Related Art.

In recent years it has become apparent that the sea offers an enormousbiomedical potential. The marine environment is quite distinct from theterrestrial environment and a vast number of marine natural productswith novel molecular architectures were already isolated and purifiedfrom diverse marine organisms. Surprisingly, only a small fraction ofthese novel compounds underwent detailed pharmacological and biologicalevaluations and only few of these were found to possess desirablebiological and physiological activities, see Scheuer, "Marine NaturalProducts, Chemical and Biological Perspectives" Academic Press, NewYork, Vols. I-V, (1978-1983); Kaul et al., Ann. Rev. Pharmacol., 26,117-142 (1986); Scheuer, Science, 248, 173-177 (1990).

The inventors have been engaged in the last 10 years in a researchendeavor that involves chemical, biochemical and cell biological studiesof marine natural products derived from Red Sea organisms. Thisinterconnected effort has already yielded what others have described as"perhaps the most stimulating compounds isolated from corals andsponges"--the latrunculins. See Kaul et al., Ann. Rev. Pharmacol., 26,117-142 (1986). The latrunculins, isolated from the Red Sea sponge,latrunculia magnifica react specifically with the actin-basedcytoskeleton and appear to be the most powerful probes currentlyavailable for the pharmacological investigation of microfilamentorganization and function, See: Spector et al., Science, 214, 493-495(1983); Coue et al., FEBS Lett., 213, 316-318 (1987) and Spector et al.,"Cell Motility and the Cytoskeleton", 13 127-144 (1989) Also see, U.S.Pat. No. 4,857,538 to Kashman et al. entitled New Compounds for theStudy and Treatment of Microfiliment Organization in cells.

Recently, two of the co-inventors, Drs. Kashman and Rudi have isolatedand purified six (6) new heterocyclic alkaloids from the purple tunicateEudistoma sp. and have elucidated their structures, See Rudi et al.,Tetrahedron Lett., 29, 6655-6656 (1988); Rudi et al., Tetrahedron Lett.,29, 3861-3862 (1988) and Rudi et al., J. Org. Chem., 54, 5331-5337(1989). The structures of these six (6) heterocyclic alkaloids (1)segoline A; (2) segoline B; (3) isosegoline A; (4) norsegoline; (5)Debromoshermilamine; and (6) Eilatin are shown in FIG. 1. The six (6)Eudistoma alkaloids have in common a fused tetracyclicbenzo-3,6-diazaphenanthroline ring system that was first identified inthe sponge metabolite amphimedine, see Schmitz et al., J. Am. Chem.Soc., 105, 4835-4837 (1983) and more recently in some other polycyclicaromatic alkaloids isolated from unrelated marine organisms includingsponges, tunicates (ascidians), and an anemone see for example, Ciminoet al., Tetrahedron, 43, 4023-4024 (1987 ); Bloor et al., J. Am. Chem.Soc., 109, 6134-6136 (1987); Cooray et al., J. Org. Chem., 53, 4619-4620(1988); Molinsky et al., J. Org. Chem., 53, 1340-1341 (1988); Kobayashiet al., J. Org. Chem., 53, 1800-1804 (1988); Kobayashi et al.,Tetrahedron Lett., 29, 1177-1180 (1988); Charyulu et al., TetrahedronLett., 30, 4201-4202 (1989); Molinski et al., J. Org. Chem., 54,4256-4259 (1989); and, Schmitz et al., J. Org. Chem., 56, 804-808(1991); Gunawardana et al., J. Am. Chem. Soc., 105, 4835 (1983); Carrollet al., J. Or. Chem., 55, 4427 (1990); and He Hay-yh et al., J. Org.Chem., 56, 5369 (1991).

Based on chemical structure, the Eudistoma alkaloids have beenclassified into 4 groups:

1. Three of the compounds display a high degree of structural similarityand were designated as Segoline A, segoline B, and Isosegoline A (segolmeans purple in Hebrew).

2. A fourth compound, lacks the imide moiety that is present in theabove three compounds and was designated Norsegoline.

3. Another compound contains a thiazinone moiety. It was designatedDebromoshermilamine A because it was found to be closely related toShermilamine, a compound previously purified from a different tunicatetrididemnum sp. found in Pago Bay, Guam, as reported by Cooray et al.,J. Org. Chem., 23, 4619-4620 (1988).

4. The last compound was designated Eilatin (from tunicate collected inEilat) is the most remarkable in having a rare, highly symmetricalheptacyclic structure.

In addition to these six (6) natural compounds, Drs. Kashman and Rudihave synthesized several derivatives of Segoline A as shown in Scheme Iat FIG. 2(A), Segoline B as shown in Scheme II at FIG. 2(B); see, Rudiet al., J. Org. Chem., 54, 5331-5337 (1989); also see, Gellerman, Rudi &Kashman, Tet. Letters, 33, 5577 (1992). Others have also attempted tosynthesize similar compounds See, Ali et al., J. Chem. Soc. Chem.Commun., 1453 (1992)

Chemically, the six (6) Eudistoma alkaloids appear to belong to agrowing class of novel marine alkaloids that have in common a fusedtetracyclic benzo-3,6-diazaphenanthroline ring system. There are scantreports concerning the biological activities of these related speciesindicating that many of them are cytotoxic to a variety of cancer celllines see Cimino et al., Tetrahedron, 43, 4023 (1987); Bloor et al., J.Am. Chem. Soc., 109, 6134 (1987); Molinski et al., J. Org. Chem., 53,1340 (1988); Kobayashi et al., J. Org. Chem., 53, 1800 (1988); Kobayashiet al., Tetrahedron Lett., 29, 1177 (1988); Charyulu et al., TetrahedronLett., 30, 4201 (1989); Molinski et al., J. Org. Chem., 54, 4256 (1989);Schmitz et al., J. Org. Chem., 56, 804 (1991) but the mechanism by whichthey exert their cytotoxic effects is completely unknown.

At present it is not even clear whether all these novel marine alkaloidsshould be classed together, and whether they are produced by the sourceorganism or by symbionts, see Rudi et al., J. Org. Chem., 54, 5331-5337(1989). The presence of six different alkaloids in the same Eudistoma sporganism is without precedent and provides a unique opportunity to shedsome light on these problems.

SUMMARY OF THE INVENTION

The present invention discloses biological applications of synthetic andnatural alkaloids derived from the tunicate Eudistoma sp. One aspect ofthe present invention is a for method regulating cell growth. The methodcomprises contacting one or more cells with an effective concentrationof a compound for regulating cell growth. These compounds include:

Segoline A, Segoline B, Isosegoline A, Norosegoline,Debromoshermilamine, Eilatin, 4-methylpyrido[2,3,4-kl]acridine,pyrido[2,3,4-kl]acridine, 1-acetyl-2,6-dimethylpyrido[2,3,4-kl]acridine,a compound having the chemical structure: ##STR1## wherein R₁ isselected from time group consisting of phenyl, halogen, hydroxy, CO₂-Methyl, HO-Methyl, COCH₃, CH₃ and H; 12-demethyl carboxylateNorsegoline, Seco Eilatin, 4,7-dinitroeilatin, a compound laving thechemical structure: ##STR2## wherein Z is selected from the groupconsisting of phenyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃,CH₃ and H; N(II)-methyl Segoline A, N(I)-dimethyl Segoline A(I⁻),N(II)-dimethyl Segoline A(I⁻), N(12)-methyl-Isogoline, N(1)-dimethyl-Norsegoline, N(8) -dimethyl Norsegoline, N(II)-methyl SegolineB, N(I) dimethyl Segoline B(I⁻), a compound having the chemicalstructure: ##STR3## and a compound having the chemical structure of:##STR4## and a compound having the chemical structure of: ##STR5## andderivatives and combinations of these compounds.

An effective concentration range for using these compounds can rangefrom approximately 0.1 μM to 100 μM. The effective concentration rangefor a Eilatin, the most potent of these compounds, is from 0.01 μM to0.99 μM, and the effective concentration range for the other compoundsof the present invention is from about 1.0 μM to 100 μM. For the cellstested in the present invention the most effective concentration wasshown to be:

(a) 12-52 μM for Segoline A, Segoline B and Isosegoline;

(b) 8-40 μM for Norosegoline;

(c) 6-32 μM for Debromoshermilamine;

(d) 0.05 to 0.5 μM for Eilatin;

(e) 7.5-12.5 μM for 4-methylpyrido[2,3,4-kl]acridine; and

(f) 2-5 μM for pyrido[2,3,4-kl]acridine.

The method of the present invention has been shown to suppress growth oftumor cells, and induced differentiation of the tumor cells.Additionally, the method has induced reverse transformation of the tumorcells.

In transformed cells, the method of the present invention has been shownto induce reverse transformation. The method of the present inventionhas been shown to inhibit the proliferation of cells. The examples showthat the method of the present invention affects cyclic AMP mediatedbiological processes. The compounds of the present invention when usedat their effective concentrations, affect the cyclic AMP mediatedbiological processes of the cells to achieve the results describedabove.

Another aspect of the present invention is the synthesis of a number ofpyridoacridines and intermediates in the synthesis of thesepyridoacridines. These compounds and their scheme for synthesis areillustrated in FIGS. 2C-2I. One compound according to the presentinvention has the chemical structure: ##STR6## in which R₁ can includethe following groups: phenyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl,COCH₃, CH₃ and H. Preferred compounds include where R₁ is CH₃ or H.

Another compound according to the present invention has a chemicalstructure: ##STR7## wherein R₁ can include the following groups: phenyl,halogen, hydroxy, CO₃ -Methyl, HO-Methyl, COCH₃, CH₃ and H and R₂ caninclude the following groups: NHOCH₃, NH₂ and N₃ ; and preferably whenR₁ is H or CH₃.

Another compound disclosed by the present invention 25 has the chemicalstructure: ##STR8## in which R₃, R₄, and R₅ can include the followinggroups: phenyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃, H; andCH₃. Preferably in which R₃ and R₅ are CH₃, and R₄ is COCH₃.

Another compound according to the present invention has a chemicalstructure: ##STR9## in which R₄ call include the following groups:penyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃, of H, and C₃.

Another compound according to the present invention has a chemicalstructure: ##STR10## in which R₆ can include the following groups:NHOCH₃, N₃ and NH₂ ; and R₇ can include the groups: H, CH₃, phenyl,halogen, hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃.

Another compound according to the present invention has the chemicalstructure: ##STR11## in which R₆ can include the following substituents:NHOCH₃, N₃ and NH₂ ; and R₇ can include: H, CH₃, phenyl, halogen,hydroxy, CO₂ -Methyl, HO-Methyl, and COCH₃.

Another compound according to the present invention has the chemicalstructure: ##STR12## in which R₈ can include the following substituents:H, phenyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃ and OCH₃ ;preferably R₈ is OCH₃.

Another compound according to the present invention has a chemicalstructure: ##STR13## in which R₈ and R₉ can include the followingsubstituents: H, CH₃ phenyl, halogen, hydroxy, CO₂ -Methyl, HO-Methyl,COCH₃ and OCH₃ ; preferably where R₈ and R⁹ are OCH₃ or H.

Other compounds according to the present invention are derivatives ofSegoline A, and Eilatin, compounds 30, 32 and 34, respectively, asillustrated in FIG. 2I, and the intermediates leading to thesecompounds: ##STR14##

Other such derivatives may be prepared from each of the compoundsdescribed above and illustrated in FIGS. 1-2(A-I), in a similar manneras synthesized in the examples according to chemical methodologies wellknown to those skilled in the art.

Another aspect of the present invention is a process for synthesizingthe compounds described above and the intermediate derived insynthesizing these compounds. The process includes the steps of:

(a) dissolving a compound having the chemical structure: ##STR15##wherein R₁ is H or CH₃, in acetic acid with m-nitrosulfonic acid sodiumsalt at a temperature of about 65°-70° C.;

(b) adding vinylphenylketone to the solution of step (a) at atemperature of about 65°-70° C.;

(c) heating the reaction mixture of step (b) to a temperature of about110° C.±5° C., or reacting in an ultrasonic bath at ambient temperature,for 1.5 hrs.±0.5 hrs.

(d) cooling the reaction mixture of step (c) to about 5° C.;

(e) contacting the reaction mixture of step (d) with ammoniacal ice,thereby forming a precipitate of a compound having the chemicalstructure: ##STR16## and, (f) separating the compound of step (e) fromthe mixture. The intermediate obtained in step (e) can then be used by:

(g) dissolving the intermediate obtained in step (e) in 80% sulfuricacid;

(h) heating the solution of step (g) to a temperature solution in anultrasonic bath at ambient temperature, for about 1.5±0.5 hrs.;

(i) cooling the reaction mixture of step (h) to about 5° C.;

(j) contacting the reaction mixture of step (i) with ammoniacal ice;

(k) extracting the resulting amine with methylene chloride; and

(l) evaporating the methylene chloride yielding the free amine compoundhaving the chemical structure: ##STR17##

In the case where the R₁ substituent of the starting compound is H theprocess also includes the step of separating the compound obtained instep (e) from its isomer, preferably by flash silica gel chromatographyutilizing CHCl₃ --CH₃ OH as the eluant. The synthesis can continue fromstep (1) by:

(m) dissolving the amine compound in 1.5N HCl;

(n) cooling the solution of step (m) to about 0° C.;

(o) adding NaNO₂ to the solution of step (n);

(p) after about 20 minutes at about 0° C. adding NaN the solution ofstep (o);

(q) after about 10 minutes adding ammonia to pH 10 thereby obtaining acrude azide compound having the chemical structure: ##STR18## (r)extracting the crude azide with a solvent; (s) evaporating the solventthereby recovering the crude azide; and

(t) purifying the crude azide to yeild the pure azide compound.Preferably, in this process the solvent is methylene chloride and thepurifying step is by silica gel chromotography. In continuing thesynthesis from step (t), the process includes:

(u) dissolving the azide in durane under argon;

(v) heating the solution (u) to 200° C. for approximately 30 minutes;

(w) cooling (v) to about 5° C.;

(x) dissolving (w) in a suitable solvent;

(y) extracting 2NHCl a pyridoacridine compound having the chemicalstructure: ##STR19## (z) neutralizing the acid with ammonia andextracting the resulting salt with a suitable solvent;

(aa) evaporating the solvent to recover the pyridoacridine ocmpound; and

(ab) purifying the resulting pyridoacridine compound. Preferably, thesolvent of step (r) is CH₂ Cl₂ --CH₃ OH and purifying step (ab) is bysilica gel chromatography.

Another process disclosed in the present invention is the synthesisincluding the steps of:

(a) combining a compound having the chemical structure ##STR20## ando-chlorobenzoic acid under Ullmann reaction conditions to afford asubstituted dephenylamine;

(b) inducing cyclization of the pyridine ring of the substituteddephenylamine in the presence of poly phosphric acid (5 eq.) and at atemperature of 125° C.±5° C., or in an ultrasonic bath at ambienttemperature, for about 1.5±0.5 hours; and;

(c) heating the reaction mixture obtained in step (b) a temperature ofabout 125° C.±5° C., or in an ultrasonic bath at ambient temperature,for about 1.5±1/2 hrs. in the presence of catalytic amounts of sulfuricacid (H₂ SO₄) to yield a compound having time chemical structure:##STR21##

The synthesis can continue from the above intermediate as follows:

(d) reducing time compound obtained in step (c) with Na(Hg) to yield1-amino-4-methylacridine. In addition, tire synthesis calm be continuedby:

(e) heating said 1-amino-4-methylacridine with acetyl acetone andcatalytic amounts of acid (H₂ SO₄) in amyl alcohol at about atemperature of about 125°±5° C., or in an ultrasonic bath at ambienttemperature, for about 1.5±0.5 hrs. thereby yielding1-acetyl-2,6-dimethylpyrido-(2,3,4-kl)acridine. The1-acetyl-2,6-dimethylpyrido (2,3,4-kl)acridine is then extracted fromthe reaction mixture.

The intermediate can be further processed as follows:

(g) reacting said 1-amino-4-methylacridine with cyclohexanone andcatalytic amounts of acid (H₂ SO₄) in amylalcohol at a temperature ofabout 125° C.±5° C. for 1.5±1/2 hrs. thereby yielding a compound havingthe chemical structure: ##STR22##

This compound can be further processed by:

(h) extracting the compound obtained ill step (g) from the reactionmixture.

If time starting materials, or intermediates of the above process aredemethylated, i. e. do not conta in a methyl group, the process wouldfinally yield 12-demethyl carboxylate Norsegoline.

Another process according to this invention is the synthesis accordingto the following steps:

(a) combining 1,8-phenathroline -5,6-dione in aniline or P-OMe-aniline(2.5 eq.) and in HOC (10 eq.),

(b) reacting the mixture under reflux conditions, or in an ultrasonicbath at ambient temperature, for about 11/2±1/2 hrs. thereby yielding acompound having the chemical structure: ##STR23## (c) extracting thecompound obtained in step (b) from the reaction mixture; and

(d) purifying the compound.

Another process for synthesis according to the present inventionincludes the following steps:

(a) combining 9,10-phenanthenequinone in aniline or P-OMe-aniline (2.5eq.) and in HOC (10 eq.),

(b) reacting the mixture under reflux conditions or in an ultrasonicbath at ambient temperature, for about 11/2±1/2 hrs. thereby yielding acompound having the chemical structure: ##STR24## (c) extracting thecompound obtained in step (b) from the reaction mixture; and

(d) purifying the compound.

The compounds of the present invention can be modified in various ways,for example nitration of Segolina A & B yielding compounds 29 and 31,and of Eilatin yielding compound 33 can be carried out, followed byreduction of the resulting nitro groups to yield the corresponding aminocompounds 30, 32 and 34. Eilatin has been demonstrated to affordSelectively the 4,7-dinitro derivative 33. Reduction of the nitro groupyields the corresponding amino groups 34 from which a variety ofderivatives are prepared (--OH, halogens, --CN, --CO₂ H, etc.) via thediazonium ion. The regioselectivity of nitration (to C-14 in case ofSegoline A and Segoline B, and C-4 and C-7 in case of Eilatin) has beendemonstrated. Hydrogenation, with H₂, dithionite or Sn or Fe/HClreduction of the nitro compounds affords the amino derivatives. Theamines are further modified via diazonium salts (NaNO₂ --HCl), and thenchanged into different groups using well established procedures forthese compounds, (e.g. halogens, --CN) other nitration reagents, such asNO₂ BF₄, HNO₃ -acetic acid, HNO₂ --H₂ SO at suitable reaction conditionsare also used to achieve other substitutents. Many of the resulting newcompounds are amenable to further chemical changes.

Another embodiment of the present invention includes a compound havingthe chemical structure: ##STR25## wherein R₁₀ is a substituent which caninclude a phenyl, halogen, hydroxy, CO₂ -Methyl, COCH₃, CH₃, H, NHOCH₃,NO₂, NH₂, or N₃. In addition, the present invention includes compoundshaving the following chemical structures: ##STR26## and derivativesthereof. Furthermore, the present invention includes a compound havingthe chemical structures selected from the group consisting of: ##STR27##wherein R₁₁ is NO.sub. 2.

Another embodiment of the present invention includes a process forsynthesizing, pyrido[k,l]acridine compounds, which includes thefollowing steps:

Reacting a first compound which can include o-benzyquinone,hydroquinone, combinations and derivatives thereof, with a secondcompound which can include kynuramine, derivatives of kynuramine andcombinations thereof, under appropriate reaction conditions, whichpreferably include mild oxidative conditions in aqueous EtOH and anoxidant which may include NaIO₃, CeCL₃ or Fe(SO₄)₂. The reaction therebyyielding a pyrido[k,l]acridine compound.

Another preferred process of the present invention is the synthesis ofeilatin and its derivatives, which includes the following steps:

Reacting a first compound having the chemical structure: ##STR28## inwhich R₁₀ is a substituent which may include a phenyl, halogen, hydroxy,CO₂ -Methyl, HO-Methyl, COCH₃, CH₃, H, NHOCH₃, NO₂, NH₂, or N₃. Thefirst compound is reacted with a second compound in a 1:2 ratio,respectively. The second compound may include kynamurine having thechemical structure: ##STR29## or derivatives of kynamurine andcombinations thereof under appropriate, mild oxidative reactionconditions.

In this process, a preferred intermediate is a1,10-phenathroline-5,6-dione derivative, which is further preferablytreated with a mild base, preferably with NH₃ --NaOH, to yield eilatinhaving the chemical structure: ##STR30##

For a better understanding of the present invention, reference is madeto the following description and examples, taken in conjunction with theaccompanying tables, the scope of which is pointed out in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Eudistoma alkaloids, Segoline A SegolineB, Isosegoline A, Norsegoline, Debromoshermilamine and Eilatin.

FIG. 2(A) shows Scheme I, the chemical transformation of Segoline A(1);

FIG. 2(B) shows Scheme II, the chemical transformation of Segoline B(2);

FIG. 2(C) shows Scheme III, the steps for synthesizing compound 9,4-methylpyrido[2,3,4-kl]acridine, and compound 10,pyrido[2,3,4-kl]acridine;

FIG. 2(D) shows Scheme IV, the steps for synthesizing compound 14,1-acetyl-2,6,-dimethylpyrido[1,3,4-kl]acridine, and compound 15,6-methylpyrido[2,3,4-kl]acridine;

FIG. 2(E) shows the structure of compound 25, 12-demethyl-carboxylateNorsegoline;

FIG. 2(F) shows the synthesis of compound 26, a seco Eilatin from1,8-phenathroline-5,6-dione;

FIG. 2(G) shows the structure of compound 27, a seco Eilatin synthesizedfrom 9,10-phenantrenequinone;

FIG. 2(H) shows the synthesis of compound 28, monodiazaeilatin (orphenylascididenine) from 5-nitro-4,5-diphenyl-phentroline via a nitreneintermediate; and

FIG. 2(I) shows Scheme V, the selective nitration of Segoline A & B andof Eilatin followed by reduction of the resulting nitro groups to yieldthe corresponding amino compound.

FIG. 3 is a series of phase-contrast micrographs showing the long-termeffects of the Eudistoma alkaloids on morphological appearance of livingmouse neuroblastoma N1E-115 cells. A,B: Untreated cells, 3 (A) and 8 (B)days after subculture. C,D: Cells grown for 3 (C) and 8 (D) days with12.8 μM Debromoshermilamine. E,F: Cells grown for 7 days withIsosegoline A (39 μM) and Norsegoline (16.3 μM), respectively.

FIG. 4 is a series of phase-contrast micrographs showing the long-termeffects of the adenylyl cyclase activator, forskolin and the Eudistomaalkaloids on morphological appearance of N1E-115 cells. A,B: Untreatedcells 3 (A) and 6 (B) days after subculture. C,D: Cells grown for 3 (C)and 6 (D) days with 50 μM forskolin. E,F: Cells grown for 3 (E) and 6(F) days with 39 μM Segoline A. G,H: Cells grown for 3 (G) and 6 (H)days with 0.14 μM Eilatin.

FIG. 5 is a series of Hoffman Modulation Contrast micrographs showingthe long-term effects of the two synthetic pyridoacridines,4-methylpyrido[2,3,4-kl]acridine and pyrido[2,3,4-kl]acridine onmorphological appearance of N1E-115 cells. Cells were grown for six daysin the absence of drugs (A), or in the presence of: 10 μM4-methylpyrido[2,3,4-kl]acridine (B), 4 μM pyrido[2,3,4-kl]acridine (C),0.08 μM Eilatin (D), 75 μM forskolin (E).

FIG. 6 is a series of phase-contrast micrographs showing the effects ofthe Eudistoma alkaloids on morphological appearance of NIL8 fibroblastcells. Cells were grown for 5 days in the absence of drugs (A), and inthe presence of 25 μM forskolin (B), 1.4 μM Eilatin (C), or 12.8 μMDebromoshermilamine (D).

FIG. 7 is a series of Hoffman Modulation Contrast micrographs showingthe effects of the two synthetic pyridoacridines,4-methylpyrido[2,3,4-kl]acridine and pyrido[2,3,4-kl]acridine onmorphological appearance of NIL8 fibroblast cells. Cells were grown for6 days in the absence of drugs (A), or in the presence of 10 μM4methylpyrido[2,3,4-kl]acridine (B), 4 μM pyrido[2,3,4-kl]acridine (C),0.08 μM Eilatin (D), or 75 μM forskolin (E).

FIG. 8 is a series of Hoffman Modulation Contrast micrographs showingthe effects of the Eudistoma alkaloids on morphological appearance ofthe HSV-transformed NIL8 fibroblasts (NIL8-HSV). A-D Cells grown for 6days in the absence of drugs (A), or in the presence of 75 μM forskolin(B), 13 μM Segoline B (C) or 0.21 μM Eilatin (D). E and F show theappearance of the Segoline B- and Eilatin-treated cells, 7 days afterremoval of the toxins.

FIG. 9 shows fluorescence micrographs of fixed NIL₈ -HSV cells stainedwith rhodamine-phalloidin to illustrate the effects of Segoline B onF-actin organization. A: F-actin staining in control cells. B: F-actinstaining in a cell treated for 4 days with 13 μM Segoline B.

FIG. 10 (A-E) is a series of Hoffman Modulation Contrast micrographsshowing the effects of the two synthetic pyridoacridines,4-methylpyrido[2,3,4-kl]acridine and pyrido[2,3,4 -kl]acridine onmorphological appearance of the virus-transformed NIL8-HSV fibroblastcells. Cells were grown for 6 days in the absence of drugs (A), or inthe presence of 10 μM 4-methylpyrido[2,3,4-kl]acridine (B), 4 μMpyrido[2,3,4-kl]acridine (C), 0.08 μM eilatin (D), or 75 μM forskolin(E).

FIG. 11 is a graph showing the effects of the Eudistoma alkaloids onpyruvate kinase activity in cultured rat hepatoma FAO cells. Enzymeactivity v/V_(max) was assayed as described in Example IV in partiallypurified extracts from cells incubated for 1 h in the absence ( ) orpresence of 100 μM CPT-cAMP (•) and 26 μM segoline A ( ) as a functionof substrate concentrations (phosphoenolpyruvate, PEP). The dashed linerepresents the K_(m) for the substrate and it was 0.14 for the control,0.52 for the cAMP analog, and 0.44 for segoline A. K_(m) 's fornorsegoline and Debromoshermilamine were 0.4 and 0.46, respectively.

FIG. 12 is a graph showing the effects of Eudistoma alkaloids on levelsof PEPCK mRNA in FTO-2B rat hepatoma cells. The cells were exposed for 4hours to serum-free medium which contained: 5 μM CPT-cAMP (lane 1), 5,26 and 52 μM segoline A (lanes 2-4), 2.6, 12.8 and 38.4 μMDebromoshermilamine (lanes 5-7) or to serum-free medium alone (lane 8).

FIG. 13 is a graph showing the effects of Debromoshermilamine on thesecretion of growth hormone (GH) from dispersed rat anterior pituitarycells. Each bar represents the mean ±s.e.m of four wells.

FIG. 14(A) shows the chemical structure of norsegoline and eilatin; andFIG. 14 (B) shows a proposed biomemetic synthesis ofpyrido[k,l]acridines, particularly the compounds norsegoline andeilatin.

FIG. 15 shows the synthesis of benzo-pyrido[k,l]acridine.

FIG. 16 shows the synthesis of compound 48, a potential precursor ofbenzo-norsegoline.

FIG. 17 shows the synthesis of 6-oxy-pyrido[k,l]acridine.

FIG. 18 shows the synthesis of dihydropyrido[k,l]acridine andpyrido[k,l]acridine.

FIG. 19 shows the synthesis of 1:2 addicts, compounds 54 and 55 aspotential precursors of eilatin.

FIG. 20 shows the synthesis of compounds 58 and 59 as potentialprecursors of eilatin.

FIG. 21 shows the synthesis of phenyl-ascididemin.

FIG. 22 shows a preferred biommetric strategy for the synthesis ofeilatin.

FIG. 23 shows the preferred two step, and three step biomemetic totalsynthesis of eilatin carried out in Example VIII.

DETAILED DESCRIPTION OF THE INVENTION

Evaluation of the biological effects of the six Eudistoma alkaloids andtwo of the synthetic pyridoacridines, 4-methylpyrido[2,3,4-kl]acridineand pyrido[2,3,4-kl]acridine revealed that all eight compounds possesspotent growth regulatory properties (see Example V). A singleapplication of the Eudistoma alkaloids or synthetic pyridoacridinesinhibited cell division in normal and cancer cell lines and induceddifferentiation in neuroblastoma cells and reverse transformation invirus-transformed fibroblast cells. Biochemical studies further showedthat the alkaloids mimic the short-term effects of cAMP analogues oncellular processes that are known to be mediated by cAMP (see ExampleVI). These results suggest that the growth regulatory properties of theEudistoma alkaloids are mediated by cAMP. However, in marked contrast tothe permanent effects induced by a single application of the Eudistomaalkaloids, a single application of cAMP analogues or agents that elevatecytosolic cAMP levels did not sustain growth inhibition. The cellsresumed division within 48 h after drug application and became confluentafter a week. These results strongly suggest that the Eudistomaalkaloids exert their growth regulatory effects via a unique and novelaction on the cAMP signaling system.

FIG. 1 shows the structure of six Endistoma alkaloids. Segoline A;Segoline B; Isosegoline A; Norsegoline; Debromoshermilamine; andEilatin. FIG. 2 (A) shows Scheme I, the chemical transformation ofSegoline A(1); FIG. 2 (B) shows Scheme II, the chemical transformationof segoline B(2); FIG. 2 (C), Scheme III shows the structure ofsynthetic pyridoacridine (9) 4-methylpyrido[2,3,4-kl]acridine; and FIG.2(D), Scheme IV shows the structure of synthetic pyridoacridine (10)pyrido[2,3,4-kl]acridine.

The inventors have surprisingly discovered that a one time exposure of acloned cell line isolated from the C-1300 mouse neuroblastoma tumor toany one of the eight (8) compounds completely inhibited cellproliferation and induced a process of differentiation during which thecells extended long neurites and developed other characteristic neuronalproperties (FIGS. 3-5). Likewise, fibroblast cells transformed byhamster sarcoma virus (HSV) which display altered growth patterns andaberrant morphologies responded to a one time exposure of the Eudistomaalkaloids (FIGS. 8,9) and of the two synthetic pyridoacridines (FIG. 10)by restoration of normal cell growth and morphology, a phenomenon knownas "reverse transformation" that is described by Lockwood et al., J.Cell. Biochem., 33, 237-255 (1987). FIGS. 6 and 7 show that in normalfibroblasts each of the eight (8) compounds slowed down cell divisionand caused cell flattening or elongation that resulted in lowersaturation densities. It should be noted, however, that there arequantitative as well as qualitative differences among the four groups ofthe Eudistoma alkaloids and between the six (6) natural compounds andthe two (2) synthetic pyridoacridines with regard to their actions atthe cellular level. For example, Eilatin, which has an unusuallysymmetrical heptacyclic structure, was found to be 100 times more potentthan the other five (5) naturally occurring compounds, as well as thesynthetic pyridoacridines, and more cytotoxic to transformed cells thanto normal cells (See example V).

The effects of the Eudistoma alkaloids and the two syntheticpyridoacridines on normal fibroblasts are strikingly similar to thoseobtained by chronic treatment with cAMP analogues or agents that elevatecAMP. In addition, many of the reagents and growth conditions thatregulate growth and differentiation of transformed cells do so byelevating intracellular cAMP levels as reported by Lockwood et al, J.Cell. Biochem., 33, 237-255 (1987); Willingham, Int. Rev. Cytol., 44,319-363 (1976); Schubert, "Developmental Biology of cultured Nerve,Muscle, and Glia", John Wiley & Sons, New York (1984). Accordingly, theinventors carried out the foregoing experiments to determine whether theEudistoma alkaloids exert their growth regulatory effects via the cAMPsignaling system. At the cell biological level the action of theEudistoma alkaloids were compared to the action of several cAMPanalogues and agents that elevate intracellular cAMP levels. Theseinclude the adenylyl cyclase activator forskolin, the phosphodiesteraseinhibitors theophylline and IBMX, the cAMP analogues dibutyryl cAMP(dbcAMP), 8-(4-chlorophrnylthio) cyclic AMP (CPT-cAMP) and8-chloro-cAMP. The results of these experiments are presented in FIGS.4-8, 10 and show that the short-term effects of the Eudistoma alkaloidsresemble those of the cAMP elevating agents. However, in marked contrastto these agents whose effects begin to wane within 48 hours after drugaddition, it took only a single exposure of the normal or thetransformed cells to the Eudistoma alkaloids to inhibit cellproliferation and to induce and sustain what appears to be permanentdifferentiation in neuroblastoma cells and reverse transformation in thevirus transformed fibroblast cells.

Particularly significant is the comparison with the two cAMP analoguesCPT-cAMP and 8-chloro-cAMP. These site-selective cAMP analogues whichbind preferentially to type II cAMP-dependent protein kinase were shownto be powerful regulators of growth and differentiation of cancer celllines and are considered as new possible tools in the treatment ofcancer as reported by Katsaros et al., FEBS Lett. 223, 97-103 (1987),Tagliaferri et al. Cancer Res. 48, 1642-1650 (1988), and Tortora et al.,Proc. Natl. Acad. Sci. USA 87, 705-708 (1990). The results described inthese Examples indicate that the Eudistoma alkaloids and the syntheticpyridoacridines may affect target proteins that are associated withactivation of type II rather than type I cAMP-dependent protein kinaseand that their pharmacological properties are more desirable than thoseof the site-selective cAMP analogues.

At the biochemical level, the inventors assessed the action of severalof the Eudistoma alkaloids, and the two synthetic pyridoacridines, ontwo well characterized short-term cAMP-mediated cellular processesinvolved in hepatic glucose metabolism, as well as on release of growthhormone from anterior pituitary cells. The cAMP-mediated processesinvolved in hepatic glucose metabolism include inhibition of pyruvatekinase (PK) activity and induction of mRNA for P-enolpyruvatecarboxykinase (PEPCK). PK is a key glycolytic enzyme whose activity isinhibited by cAMP- dependent phosphorylation, whereas PEPCK is a keyenzyme in the gluconeogenesis pathway whose gene transcription rate isstimulated by cAMP, see Pilkis et al., Annu. Rev. Nutr., 11, 465-515(1991); Maghrabi et al., J. Biol. Chem., 257, 233 (1982); Hod et al., J.Biol. Chem. 259, 15603-15608 (1984); Granner et al., J. Biol. Chem.,265, 10173-10176 (1990). Growth hormone is a major product of anteriorpituitary cells whose release is regulated by cAMP, see Bilezikjian etal., Endocrinology, 113, 1726-1731 (1983); Ray et al., Endocrinology 45,175-182 (1986); Gabriel et al., Neuroendocrinology, 50, 170-176 (1989).The inventors have found that the Eudistoma alkaloids, like cAMPanalogues, inhibit PK activity and stimulate PEPCK mRNA accumulation, aswell as growth hormone release (FIGS. 11-13) suggesting that the growthregulatory effects of the Eudistoma alkaloids are mediated by the cAMPsignaling system.

Major Implications of The Present Invention

1. By using a cell line isolated from the C-1300 mouse neuroblastomatumor and virus transformed fibroblast cells the inventors have foundthat the Eudistoma alkaloids and the two synthetic pyridoacridinesregulate cellular growth and differentiation of cancer cell lines.Furthermore, by using the normal counterpart of the transformedfibroblast cell line, the inventors found that the effects of thealkaloids on normal cells are similar to those of cAMP analogues oragents that elevate cAMP. Normal cells, however, are less sensitive tothese alkaloids and in particular to the most potent compound, Eilatin.In fact, Eilatin has no effect on normal cells at concentrations thatinduce differentiation and reverse transformation of transformed cells.

The cell culture systems described above are highly suitable forstudying the effects of novel drugs on the regulation of cellular growthand differentiation. Neuroblastoma cell lines have been used extensivelyas a model of nerve cell differentiation because various agents andtreatments which inhibit cell proliferation cause neuroblastoma cells toundergo a controlled process of differentiation during which they extendlong neurites and develop other characteristic neuronal properties; forexample see: Schubert, "Developmental Biology of cultured Nerve, Muscle,and Glia", John Wiley & Sons, New York (1984). Likewise, manyvirus-transformed cells which display altered growth patterns andaberrant morphologies can respond to various agents, primarily thosethat elevate intracellular cAMP levels, by restoration of normal cellgrowth and morphology, a phenomenon known as "reverse transformation",as described by Lockwood et al., J. Cell. Biochem. 33 237-255 (1987).Finally, the concomitant use of normal fibroblast cells is highlysignificant in better characterizing the biological effects of noveldrugs and their relationship to other compounds with known biologicalactions. Moreover, the use of normal cells helps to determine whether agiven compound is simply cytotoxic, and indiscriminately affects normaland transformed cells, or whether the compound possess desirablepharmacological properties that can be further exploited in developingnew drug.

2. Another important implication of the present invention concerns thefindings that the Eudistoma alkaloids may act on the cAMP signalingsystem. When compared to other drugs that are currently available toprobe the cAMP system, or for that matter the other signal transductionsystems that are involved in growth regulation of cancer cells, theEudistoma alkaloids have a unique mode of action. This aspect of thepresent invention potentially opens new avenues in the ever expandingfield of cAMP research and in the development of new drugs tocharacterize and correct perturbations of this ubiquitous centralregulatory system, and in particular perturbations in normal cell growthand differentiation that lead to cancer.

The cAMP system functions to coordinate and regulate diverse cellularprocesses ranging from breakdown of glycogen in liver cells, toregulation of growth and differentiation, see Stryer, "Biochemistry" Ch.38, W. H. Freeman & Co, New York (1988); Alberts et al., "MolecularBiology of the Cell"2d ed., CH. 12, Garland Publishing, New York (1989);Willingham, (1976) supra; Schubert, (1984) supra; and Tortora, et al.,Proc. Natl. Acad. Sci. USA, 87 705-708 (1990). Perturbations of thissystem are responsible for many disease states, and may underlieoncogenic transformation of normal cells, as discussed by Lockwood etal., (1987) supra. Although cyclic AMP is considered to be the mostimportant regulatory molecule in mammalian cells, the mechanisms bywhich it exerts its diverse effects are far from being understood. Thisis due to the complex multistep and pleitropic process by which bindingof hormones to plasma membrane receptors stimulate the membrane boundenzyme adenylyl cyclase which catalyzes cAMP synthesis. Cyclic AMP inturn activates the cAMP-dependent protein kinases which cause a cascadeof protein phosphorylations, and simultaneously alters numerous steps inmany metabolic pathways. Termination of hormonal action is achieved bycyclic nucleotide phosphodiesterases enzymes which degrade cAMP, and byphosphoprotein phosphatases, enzymes which dephosphorylate the targetproteins. These features makes the study of the cAMP system difficultand present a major challenge for present day molecular and cellbiologists.

A fundamental approach to investigate the cyclic AMP-mediated cellularprocesses and their perturbations is to use specific drugs that willreact with one of the steps or the components involved in thecAMP-induced regulatory cascade and will affect cell behavior, asdescribed by Stryer "Biochemistry" W. H. Freeman & Co. New York, Ch. 38(1988). Thus, most of the information concerning the involvement of thecAMP signaling system in cellular activities relied on the use of drugsthat increase cytosolic cAMP levels, or mimic its action. These includethe adenylyl cyclase activator, forskolin, various methylxanthines thatinhibit cAMP phosphodiesterases, and permeant cAMP analogues. The thirstfor drugs that affect the cAMP system is manifest by the fact that sinceits discovery in 1981 as a result of a Hoeschst program designed toscreen plant extracts in India for cardiovascular and otherpharmacological activities, forskolin rapidly has become the mostextensively used drug in cAMP research, as discussed by Seamon et al.,Advances in Cyclic Nucleotide and protein phosphorylation Res., 20 1-150(1986) and is discussed in more than 500 publications per year. Theinventors' findings that the Eudistoma alkaloids and the two syntheticpyridoacridines exert a novel action on the cAMP system and are morepowerful than any of the drugs that are currently available to probethis system is a major contribution to the ever expanding field of cAMPresearch.

3. Finally, the inventors' discovery that the Eudistoma alkaloids andsynthetic pyridoacridines regulate growth and promote differentiation oftransformed cells is highly significant in the continuous search forbetter ways of restraining the uncontrolled proliferation of cancercells. The fact that similar compounds were found in different organismsin different geographic locations may indicate an important new familyof marine metabolites that regulate cell growth and differentiation.Normal cell division is a highly regulated process that is governed by acomplex set of controls, so that each stem cell generates one daughterstem cell and one cell that is committed to terminal differentiationafter a strictly limited number of division cycles. The two types ofaberration that can give rise to the unrestrained proliferationcharacteristic of cancer involve either a failure of stem cells toproduce a non-stem-cell daughter, or a failure of daughter cells todifferentiate normally, Alberts et al., "Molecular Biology Of The Cell",Garland Publishing New York, 2d ed., Ch. 21, (1989). Mutations orepigenetic changes that block the normal maturation of cells arepresumed to play a critical role in many cancers. For example,neuroblastoma and several forms of leukemia seem to arise from adisruption of the normal program of differentiation, so that thecommitted nerve or blood cell continues to divide indefinitely insteadof terminally differentiating. This is why in the treatment of cancer,drugs that promote cell differentiation like the Eudistoma alkaloids canbe very valuable therapeutic tools in what is known as "differentiationtherapy" and may be as important as currently used drugs that simplykill dividing cells.

Accordingly, the results obtained in the foregoing Examples clearly showthat the Eudistoma alkaloids and the synthetic pyridoacridines representa new class of powerful agents that regulate cellular growth anddifferentiation. These results support the contention that thesecompounds act on the cAMP signaling system in a unique and novel way.They may, therefore, have enormous potential as a new tool for cAMPresearch, as new drugs in cancer treatment, and in other treatmentsaffecting perturbations of the CAMP system.

EXAMPLES Example I

Chemical Isolation and Purification

During the course of scuba diving expeditions to survey the constituentsof tunicates in the Red Sea, the inventors collected samples of thepurple tunicate Eudistoma sp. from various locations in the Gulf ofEilat, Straits of Tiran, and Gulf of Suez. The samples were deep-frozenimmediately after collection, freeze-dried, and then extracted withmethanol:chloroform (2:8) solution to yield ca. 2 g extract from 100 gof dry tunicate. The crude extract was separated by chromatography on asilica gel column and eluted with chloroform-hexane (7:3), chloroform,and chloroform with increasing amounts of methanol up to 15%. Repeatedchromatographies on silica gel yielded six (6) compounds. Threeadditional compounds were detected but in minuscule amounts that did notallow structural determination.

Example II

Structural elucidation of the six Eudistoma alkaloids

FIG. 1 shows the structures of the six Eudistoma alkaloids, Segoline A;Segoline B; Isosegoline A; Norsegoline; Debromoshermilamine; andEilatin, which are characterized as follows:

Segaline A (C₂₃ H₁₉ N₃ O₃, m/e 385). The most prevalent compound wasisolated in ca. 0.4% dry weight. Intensive 1D and 2D NMR studies,including Homonuclear correlation spectroscopy (COSY), differencenuclear Overhauser effects (d-NOE), short- and long-rangeCH-correlations, correlation spectroscopy via long-range coupling(COLOC) and heteronuclear COSY (HETCOSY) experiments, led to thestructural determination of the aromatic portion of the molecule and ofseveral aliphatic moieties. However, the large number of long-range CH-correlations interfered with the unraveling of the precise structure.This was resolved by a single-crystal X-ray analysis. Segoline A wasfound to be an alkaloid with a new skeleton. It embodies a tetracyclicaromatic ring system similar to that of several marine alkaloidsreported recently Schmitz et al., J. Org. Chem. 56 804-808 (1991) butthe aliphatic site and its combination with the heterocycle arealtogether new.

Segoline B (C₂₃ H₁₉ N₃ O₃, m/e 385). Isolated in ca. 0.1% dry weight,its structure was found to be closely related to that of segoline A, asevidenced by the almost identical NMR data for the great part of the twomolecules. The major differences between the NMR data of compounds 1 and2 were in the alicyclic portion of the molecule, suggesting thatcompound 2 is a diastereomer of compound 1. By measuring the CD Cottoneffects, it was concluded that Segoline B differs from Segoline A in thechirality of C-16.

Isosegoline A (C₂₃ H₁₉ N₃ O₃, m/e 385). Another more polar isomer wasisolated in ca. 0.01% dry weight. The NMR data suggested the sametri-substituted benzo-3,6-diazaphenanthroline ring system as in segolineA and segoline B, but a different imide moiety. From the NMR studies andthe chemical behavior of the molecule it was concluded that theglutarimide ring of segoline A and segoline B is replaced in IsosegolineA by a succinimide. The absolute configuration of Isosegoline A wasrelated to that of Segoline A on the basis of similar CD Cotton effects.

Norsegoline (C₁₈ H₁₄ N₂ O₃, m/e 306). A less polar compound of adifferent molecular formula was obtained in minute amounts (ca. 0.001%dry weight). From the NMR data it was evident that norsegoline containsthe same benzodiazaphenanthroline heterocycle as in compounds 1-3, butlacks the other rings. Instead, the spectroscopic data proposed acarbomethoxy moiety and another methoxy group, the location of whichwere established mainly by COSY and d-NOE experiments.

Debromoshermilamine (CS₂₁ H₁₈ N₄ O₂ S, m/e 390). Several of thecollections of the Eudistoma sp. resulted in small amounts of compound 5(ca. 0.05% dry weight). Intensive NMR work revealed this compound to bevery similar to Shermilamine A which was isolated from the purpletunicate Trididemnum sp. collected in Pago Bay, Guam by Scheuer's group,Cooray et al., J. Org. Chem., 53, 4619-4620 (1988). The only differencesbetween the two compounds is the absence of the bromine in compound 5which must, therefore, be 6-Debromoshermilamine A. More recently,Scheuer's group reported the isolation from the same tunicate of thedebromo- compound which they designated as Shermilamine B Carroll etal., J. Org. Chem., 54, 4231-4232 (1989).

Eilatin (C₂₄ H₁₂ N₄, m/e 356). The sixth compound was isolated in minuteamounts (ca. 0.001% dry weight) from a collection in Eilat, hence thename. The molecular formula revealed 21 degrees of unsaturationsuggesting a polyheterocyclic system. The high symmetry of the moleculeand the overlapping signals in the NMR spectrum hampered an unequivocaldetermination of the structure of the molecule. The structure wasfinally determined by a single-crystal X-ray analysis and the completeNMR line assignment was then carried out. Eilatin was found to possess acompletely new, highly symmetrical, seven membered ring system.

Example III

Derivatization and Chemical Transformation

As a first step towards a structure-activity study, we have prepared 19derivatives. Eight Segoline A derivatives were prepared, as shown inScheme I, FIG. 2(A). The nitration (leading to an importantintermediate), of the anisole ring was clear from the disappearance ofthe singlet at d 7.47s and in compound 22 of the OMe too. Eightderivatives of Segoline B were prepared as shown in Scheme II, FIG.2(B). N(12)-methyl-Isosegoline was prepared using diazomethane,N(1),N(8)-dimethyl-norsegoline was prepared using CH₃ I, and4,7-dinitroeilatin was synthesized by nitration of Eilatin(Disappearance of the doublet at δ=8.7Od(J=8), and shift of H-3 toδ=8.78; the other peaks are δ=9.29d(J=5.7), 8.50d(J=5.7),8.23dd(J=7.9,1.2), 7.90t(7.9).

Example IV

Synthesis of Pyridoacridines, (Compounds 9 & 10)

The synthesis of compounds 2-10 is illustrated in Scheme III, FIG. 2(c).

I. Synthesis of 4-methyl pyrido[2,3,4-kl]acridine (9, R₁ =Me)

Synthesis of Compound 3

Compound 1 described by Schatten, Org. Syn., 11, 32 (1931), (0.5 g) wasdissolved in acetic acid (30 ml) with m-nitrosulfonic acid sodium salt(1 g) at 65°-70° C. Vinylphenylketone (0.45 g) was added dropwise at65°-70° C. After 15 min the temperature was raised to 110° C. and keptat this temp. for 1.5 hrs. After cooling the reaction mixture was pouredon ice cold ammonia and the precipitate 3 filtered under vacuum.(h=25%). [Skraup reaction].

Synthesis of Compound 5

Acetamide 3 (200 mg) was dissolved in 80% sulfuric acid (5 ml) andwarmed to 130° C. for 2 hrs. The cooled solution was poured overammoniacal ice and the resulting amine 5 extracted with methylenechloride. Evaporation of the CH₂ Cl₂ gave the free amine 5 as an oil.(h=70%).

Synthesis of 7

Compound 5 (400 mg) was dissolved in 1.5N HCl (5 ml) then cooled down to0° C. NaNO₂ (150 mg) was added, and after 20 min. at 0° C., NaN₃ (150mg) was added. 10 min. later ammonia was added to pH=10 and theresulting azide extracted with CH₂ Cl₂ (3×50 ml). Evaporation of the CH₂Cl₂ yielded the crude azide 7 as dark red oil which was purified bysilica gel chromatogrphy to yield 160 mg.

Synthesis of Compound 9

As illustrated in Scheme III, FIG. 2(C) Azide 7 (200 mg) was dissolvedin durane (5 ml) under argon and slowly warmed up to 200° C. (30 min).The cooled reaction mixture was dissolved in CH₂ Cl₂ (50 ml) and 9extracted with 2N HCl (3×20 ml). The acid was then basified with ammoniaand extracted with CH₂ Cl₂ -MeOH (95:5, 3×30 ml). Evaporation followedby silica gel chromatography yielded compound 9 (100 mg).

Compound 3 Amorphous powder, m.p. 214° (acetone), C₁₈ H₁₆ N₂ O (m/z276), n_(max) : 1645, 1600, 1510, 1380, 1010, 950 cm⁻¹. d_(H) ; (CDCl₃):8.86d (J=4.2), 7.72d(J=7.7), 7.35d(J=7.7), 7.48m, 7.32m(5H),7.16d(J=4.2), 6.84brs (NH), 2.81s (Me), 1.45s(Me). d_(c) (CDCl₃): 169.5,156.1, 147.6, 144.1, 140.6, 138.7, 135.2, 127.2, 126.9, 126.5, 124.3,120.6, 112.2, 24.3, 19.1.

Compound 5 Amorphous powder, C₁₆ H₁₄ N₂ (m/z 234). d_(H) (CDCl₃): 8.74d(J=4.3 Hz), 7.27m(6H), 6.96d(J=7.6), 2.62s (Me). d_(c) (CDCl₃): 154.0,143.2, 142.7, 140.8, 136.7, 135.1, 131.7, 126.6, 126.4, 125.9, 123.8,119.3, 114.2, 22.9s (Me).

Compound 7 Red oil, C₁₆ H₁₂ N₄ (m/z 232, M-N₂), n_(max) 2210, 1620,1600, 1450, 1310, 1210 cm⁻¹. d_(H) (CDCl₃): 8.91d, (4.4 Hz),7.60d(J=7.8), 7.15m(7H) 2.82s(Me). d_(c) (CDCl₃): 157.2, 148.1, 146.2,141.7, 139.6, 136.6, 136.3, 126.1., 125.7, 124.9, 121.4, 119.6, 114.9,23.8(Me).

Compound 9 Amorphous powder, mp>300° C. C₁₆ H₁₂ N₂ (msci, m/z 233, MH⁺),n_(max) 3250, 1615, 1520, 1220, 1080, 870 cm₋₁. d_(H) (d₆ -DMSO):8.55d(J=4.8), 7.35t(^(J=) 8.0), 7.34d(^(J=) 8.0), 7.97d (J=8.0),6.95t(J=8.0), 6.98d(J=8.0), 7.42d (J=4.8), 6.82d (J=8.0), 2.4s(Me),10.4s (NH). d_(c) (d₆ -DMSO): 150.2, 147.0, 140.4, 139.9, 136.6, 132.0,131.1, 124.2, 121.3, 120.4, 118.2, 115.9, 115.6, 106.2, 102.7, 13.7.

II. Synthesis of pyrido[2,3,4-kl]acridine 10 (R=H)

As illustrated in Scheme III, FIG. 2 (C), the same synthesis as Compound9 was followed. Starting material 2, as described by Heidelberger, J.Am. Chem. Soc., 39, 1448 (1917), was reacted in the Skraup reaction,with vinylphenylketone to give a mixture of 4 and an undesired isomer inratio of 1:4. Compound 4 was separated from its isomer by flash silicagel chromatography (CHCl₃ --MeOH).

Compound 4 Amorphous powder, C₁₇ H₁₄ N₂ O (m/z 262), n_(max) 1645, 1600,1485, 1000, 980 cm⁻¹. d_(H) (CDCl₃): 8.85d, (J=4.2 Hz), 7.96d(8.0),7.70d(8.0), 7.4m(6H), 7.19d(4.2), 1.42s(Me). d_(c) (CDCl₃): 169.5,148.1, 146.5, 138.9, 137.3, 129.1, 128.9, 128.2, 126.4, 125.7, 124.0,123.6, 123.1, 114.8, 24.1(Me).

Compound 6 An oil, C₁₅ H₁₂ N₂ (m/z 220), d_(H) (CDCl₃): 8.78d(4.3),7.59t (8.2), 7.40m(6H), 7.02d(4.3), 6.63d(8.2). d_(c) (CDCl₃ : 153.4,140.8, 143.1, 141.6, 134.6, 134.1, 131.6, 126.8, 126.2, 124.7, 120.7,118.1, 115.1.

Compound 8 Dark red oil, C₁₅ H₁₀ N₄ (m/z 218, M-N₂), n_(max) 2112, 1620,1600, 1470, 1220, 1050 cm⁻¹. d_(H) (CDCl₃): 8.89d(4.2), 7.99d(8.2),7,71t(8.2), 7.40m(6H), 7.21d(4.2). d_(c) (CDCl₃): 156.8, 146.6, 145.0,140.9, 138.9, 137.0, 135.8, 126.2, 125.3, 125.0, 121.9, 120.1, 114.7.

Compound 10 Amorphous powder, mp>300° C. (d), C₁₅ H₁₀ N₂ (m/z 218) d_(H)(d₆ -DMSO): 8.46d(4.8), 7.49t(8.0), 7.58t(8.0), 8.08d(8.0), 7.06t(7.6),7.17d(8.0), 7.13d(8.0), 7.48d(4.8), 6.82d(7.8), 10.5brs. d_(c) (d₆-DMSO): 149.0, 146.0, 142.05, 140.0, 138.5, 133.0, 132.0, 125.0, 121.8,118.5, 116.5, 116.0, 112.6, 106.0, 104.4.

III. ALTERNATIVE SYNTHESIS OF PYRIDO[2,3,4,-kl]ACRIDINES

An alternative synthesis of the pyridoacridines illustrated in SchemeIV, FIG. 2(D) started from compound 11. Compound 11 was prepared in twosteps from compound 1 and o-chlorobenzoic acid, that is via an Ullmannreaction to afford a substituted diphenylamine, followed by acidcatalyzed cyclization of the pyridine ring of 11 (polyphosphoric acid,125° C., 1h).

Compound 11 was reduced with Na(Hg) to 1-amino-4-methylacridine 13Heating compound 13 with acetylacetone and catalytic amounts of acidgave pure adduct 14, upon chromatography. Specifically, compound 14 wasobtained by warming of compound 13 with acetylacetone in amylalcoholwith traces of H₂ SO₄ at 130° C. for 1.5 hr. The structure of compound14 C₁₉ H₁₆ N₂ O (eims, m/z 288, 100%)1-acetyl-2,6-dimethylpyrido[2,3,4-k,l]acridine was elucidated mainlyfrom its NMR data, by comparison of the chemical shifts with those ofother pyridoacridines as described by He Hay-Yin et al., J.O.C., 56,5369 (1991). Reacting compound 13 with cyclohexanone, instead ofacetylacetone, gave compound 15. Specifically, compound 15 was obtainedfrom cyclohexanone and compound 13 under the same conditions as compound14.

Compound 14 orange oil, C₁₉ H₁₆ N₂₀ (m/z 288), n_(max) 1680^(cm-1),d_(H) (CDCl₃) 7.59brs(NH), 7.52d(J=8 Hz), 7.36d(J=8.0), 7.30t(J=8.0),7.28d(j=8.0), 7.01d(J=8.0), 6.89t (J=8.0), 2.53s, 2.48s,2.26s. d_(c)208.7s,154.3s, 147.3s, 140.5s, 135.8s, 134.2s, 133.2d, 131.6d, 127.7d,124.1s, 121.2d, 116.3s, 116.2d, 116.0s, 115.8d, 110.8s, 95.7s, 32.4q,23.6q, 16.4q.

Compound 15, orange oil; C₂₀ H₁₈ N₂ (m/z), d_(H) (CDCl₃): 8.05d(J=8.2),7.29m, 6.94m, 3.11t (J=6.0), 3.04t(J=6.0), 2.36s, 1.89m, 1.66m.

In a similar way the inventors also synthesized 12-demethyl-carboxylateNorsegoline, compound 25 as illustrated in FIG. 2(E);n.m.r.:3.98s(OCH₃), 8.70d(J=4.6), 6.8-8.0m, 8.5br(NH).

In other syntheses illustrated in FIG. 2(f) the inventors used1,8-phenanthroline-5,6-dione, as described by Gillard et al., J. Chem.Soc., (A), 1447-1459 (1970) to synthesize a series of seco Eilatins 26:m/e 420 (100%); δ=9.61 dd, 9.25 dd, 9.14dd, 8.32d, 8-7.0m, 3.76s, 3.59s.

In the same way starting from 9,10-phenantrenequinone the inventorssynthesized 27 as shown in FIG. 2(G); m/e 418(100%); δ=9.30dd, 8.79d,8.63dd, 8.31d, 8.03d, 7.91-7.30m, 3.66s, 3.61s.

Another synthesis illustrated in FIG. 2(H) was performed leading tosynthetic monodeazaeilatin, compound 28 (or phenylascididemine, asdescribed by Kobayashi et al., Tetrahedron Lett., 29, 1177-1180 (1988))from 5-nitro-4,5-diphenyl-phenantroline, via a nitrene intermediate, asdescribed by Wentrup, "Reactive Molecules", 4, 162-264, John Wiley, NewYork (1984); and Cadogen, Quart. Rev. Chem. Soc. London, 2.2, 222-251(1968). Compound 28, δ=8.80d(J=5.0), 8.70d(5.0),7.73d(8.0),7.43m, 7.20m,6.90m, 6.63s (1H).

IV. Nitration and hydrogenation of Eudistoma Alkaloids

As illustrated in Scheme V, FIG. 2(I) nitration of Segolina A & Byielding compound 29, 31 and of Eilatin yielding compound 33 is carriedout, followed by reduction of the resulting nitro groups to yield thecorresponding amino compounds 30, 32 and 34. For example, Eilatin hasbeen demonstrated to afford selectively the 4,7-dinitro derivative 33.Reduction of the nitro group yields the corresponding amino groups 34from which a variety of derivatives are prepared (--OH, halogens, --CN,--CO₂ H, etc.) via the diazonium ion. The regioselectivity of nitration(to C-14 in case of Segoline A and Segoline B, and C-4 and C-7 in caseof Eilatin) has already been demonstrated. Hydrogenation, with H₂,dithionite or Sn or Fe/HCl reduction of the nitro compounds affords theamino derivatives. The amines are further modified via diazonium salts(NaNO₂ --HCl), and then changed into different groups using wellestablished procedures for these compounds, (e.g. halogens, --CN) othernitration reagents, such as NO₂ BF₄, HNO₃ -acetic acid, HNO₃ --H₂ SO₄ atsuitable reaction conditions are also used to achieve othersubstitutents. Many of the resulting new compounds are amenable tofurther chemical changes.

Example V

Cell Biological Studies

A. Effects of the Eudistoma alkaloids and synthetic pyridoacridines onneuroblastoma cells.

Experiments to evaluate the biological activities of the six (6) novelEudistoma alkaloids and the two (2) synthetic pyridoacridines,4-methylpyrido[2,3,4-kl]acridine and pyrido[2,3,4-kl]acridine, werefirst carried out on cells of the mouse C1300 neuroblastoma cloneN1E-115 supplied by Dr. Nirenberg, see Amano T., Richelson, E., andNirenberg M. Proc. Natl. Acad. Sci. USA, 63, 258-263 (1972). Theinventors chose the neuroblastoma N1E-115 cells because thesetumor-derived cells can be induced to express characteristic neuronalproperties by a variety of procedures see Spector, "Electrophysiology ofclonal nerve cell lines. In: Excitable Cells in Tissue Culture", Nelsonet al. eds., Plenum, New York 247-277 (1981) and can serve as a usefulexperimental system in which to explore the effects of the Eudistomaalkaloids on the biological properties of both cancer cells and neuronalcells.

The N1E-115 cells were exposed to various concentrations of the six (6)natural products shown in FIG. 1, and of the two syntheticpyridoacridines shown in Scheme III of FIG. 2. All the compoundscompletely inhibited cell proliferation and induced neuronaldifferentiation. FIG. 3 is a series of phase-contrast micrographsshowing the long-term effects of the Eudistoma alkaloids onmorphological appearance of living mouse neuroblastoma N1E-115 cells.A,B: Untreated cells, 3 (A) and 8 (B) days after subculture. C,D: Cellsgrown for 3 (C) and (8) days with 12.8 μM Debromoshermilamine. E,F:Cells grown for 7 days with Isosegoline A (39 μM) and Norsegoline (16.3μM), respectively. FIG. 5 illustrates the appearance of N1E-115 cells 6days after replating in the absence (A) and in the presence of 10 μM4-methylpyrido[2,3,4-kl]acridine (B), or 4 μM pyrido[2,3,4-kl]acridine.While the untreated cells continued to divide and reached confluence,the treated cells stopped dividing, and their appearance changeddramatically. They flattened onto the surface, increased considerably insize, and extended long neurites to resemble mature neurons. Theseeffects were not readily reversible and following removal of the drugscell division did not recommence for more than a week and the cellsmaintained their differentiated morphology. For each of the eight (8)compounds a range of concentrations was determined that inhibited cellproliferation and induced morphological differentiation (defined asenlargement of cells and outgrowth of neurites).

The range of effective concentrations for Segoline A, Segoline B andIsosegoline A was 12-52 μM, for Norsegoline and Debromoshermilamine itwas 8-40 μM and 6-32 μM, respectively, and for Eilatin which was themost potent of the compounds, it was 0.05-0.5 μM. The range of effectiveconcentrations for the two synthetic pyridoacridines,4-methylpyrido[2,3,4-kl]acridine and pyrido[2,3,4-kl]acridine was7.5-12.5 μM and 2-5 μM, respectively. In each case exposure of N1E-115cells to concentrations below this range was without a noticeableeffect, and above this range the compounds were cytotoxic. Within theeffective range the degree of inhibition of cell division was dependenton the concentration used. At higher concentrations cell division wascompletely halted and some cell death was observed. At lowerconcentrations cell division continued, but at a much slower rate.

Although all eight (8) alkaloids inhibited cell proliferation and causedprofound alterations in cell morphology, there were interestingdifferences between the alkaloids in regard to their effects on cellappearance. Thus, Debromoshermilamine treatment gave rise to a largeproportion of very flat cells of considerable size and relatively fewneurites, norsegoline induced relatively small cell bodies and numerouslong neurites, Segoline A and B, Isosegoline A, Eilatin andpyrido[2,3,4-kl]acridine induced flattened cell bodies as well asnumerous long neurites, and with 4-methylpyrido[2,3,4-kl]acridine mostneurites were short. These qualitative differences raise the possibilitythat the eight (8) alkaloids do not exert their effects on cell growthand differentiation via a common mode of action.

B. Comparison with agents that mimic or elevate cAMP.

Since most treatments that inhibit cell proliferation and inducemorphological differentiation in neuroblastoma cells involve a rise inintracellular cAMP levels, as reviewed by Schubert, "DevelopmentalBiology of cultured Nerve, Muscle and Glia", Ch 3 pp 72-165, John Wiley& Sons, New York (1984), the effects of the Eudistoma alkaloids werecompared to those of extensively used agents that mimic cAMP or elevateits levels. This comparison showed short-term similarities, but revealedstriking long-term differences between the Eudistoma alkaloids and thecAMP-related agents. These differences are illustrated in FIG. 4 whichcompares the effects of the adenylyl cyclase activator forskolin tothose of Segoline A, and Eilatin. FIG. 4 is a series of phase-contrastmicrographs showing the long-term effects of forskolin and the Eudistomaalkaloids on morphological appearance of N1E-115 cells. A,B: Untreatedcells 3 (A) and 6 (B) days after subculture. C,D: Cells grown for 3 (C)and 6 (D) days with 50 μM forskolin. E,F: Cells grown for 3 (E) and 6(F) days with 39 μ M Segoline A. G,H: Cells grown for 3 (G) and 6 (H)days with 0.14 μM Eilatin. As shown in the upper panel of FIG. 4, cellsthat have been exposed for 3 days to either 50 μM forskolin (C), 39 μMSegoline A (E) or 0.14 μM Eilatin (G), appeared remarkably similar. Allthree treatments inhibited cell division and induced morphologicaldifferentiation. However, with time in culture, the effects of a singleforskolin application waned, the cells resumed cell division and, asshown in FIG. 4 at (D) after 6 days incubation, became confluent likethe control cells (B). In contrast, a single application of Segoline Aor Eilatin induced a dramatic transition to a more homogeneous state inwhich cell division is completely blocked and, as shown in FIG. 4 at(F,H), after 6 days the cells assumed the characteristic appearance ofmature neurons.

The transient effects of forskolin on cell growth and differentiationwere also obtained with a single application of 1 mM dbcAMP plus 1 mMtheophylline, and with the potent site-selective cAMP analogues 8-chlorocAMP (250 μM) and CPT-cAMP (250-500 μM). The inventors' finding that thefully morphologically differentiated state can be reached and maintainedby a single application of the Eudistoma alkaloids suggests that thesecompounds act on the cAMP signaling system in a different manner thanthat of cAMP whose levels must remain continuously high to sustaininhibition of cell division and neurite outgrowth.

C. Effects of the Eudistoma Alkaloids on growth and morphology of normaland virus-transformed fibroblasts.

To obtain further evidence for the involvement of the cAMP signalingsystem in the growth regulatory properties of the Eudistoma alkaloids,the effects of the eight (8) alkaloids were tested on growth andmorphology of the normal hamster fibroblast NIL8 cell line and ofNILS-HSV cells, a derivative of NIL8 cells that has been transformed byhamster sarcoma virus see Hynes et al., Cell, 13, 151-163 (1978). Asreviewed by Willingham, Int. Rev. Cytology, 44, 319-363 (1976) normalfibroblasts respond to cAMP elevation by showing slower growth rates,lowered saturation densities and by becoming flatter and more elongatedthan usual. Transformed fibroblasts which display altered growthpatterns and aberrant morphologies respond to cAMP elevation byrestoration of many normal aspects of growth and morphology, aphenomenon known as "cAMP-mediated reverse transformation" as describedby Lockwood et al., J. Cell. Biochem., 33, 237-255 (1987).

FIG. 6 is a series of phase-contrast and FIG. 7 is a series of HoffmanModulation Contrast micrographs showing the long-term effects of theEudistoma alkaloids and forskolin on morphological appearance of NIL8cells. In FIG. 6 NIL8 cells were grown for 5 days in the absence ofdrugs (A), or in the presence of 25 μM forskolin (B), 1.4 μM Eilatin (C)or 12.8 μM Debromoshermilamine (D). As illustrated, a single applicationof 1.4 μM Eilatin or 12.8 μM Debromoshermilamine to NIL8 cells producedeffects which are strikingly similar to those obtained by chronicallytreating time cells with agents that elevate or mimic cAMP. The cellsincreased considerably in size, became more elongated (C) or flatter (D)than usual, their growth was inhibited and because of their large sizethey reached very low saturation densities. It should also be noted thatthe concentration of Eilatin used (1.4 μM) was toxic to both thetransformed cell lines indicating that the normal cells are lesssensitive to this alkaloid. FIG. 6B shows that the effects of a singleforskolin application were transient such that after 5 days the cellsreached the high saturation densities typical of untreated cultures (A).As shown in FIG. 7 NIL8 cells were grown for six days in the absence ofdrugs (A), or in the presence of 10 μM 4-methylpyrido[2,3,4-kl]acridine(B), 4 μM pyrido[2,3,4-kl]acridine (C), 0.08 μM Eilatin (D), or 75 μMforskolin (E). As illustrated in this figure a single application of thetwo synthetic pyridoacridines 4methylpyrido [2,3,4-kl ]acridine andpyrido [2,3,4-kl]acridine produced effects which were similar to thoseproduced by the natural alkaloids, whereas a single application of 0.08μM Eilatin, a concentration that produced differentiation in N1E-115cells and (FIG. 5 at D), and reverse transformation in NIL8-HSV cells(see below FIG. 10 at D) had no effects on NIL8 cells. This predilectiontowards cancer cells is a very desirable property in developingpotential drugs for the treatment of cancer.

FIG. 8 is a series of Hoffman Modulation Contrast micrographs showingthe effects of time Eudistoma alkaloids on morphological appearance ofthe HSV- trans formed NIL8 fibroblasts (NIL8-HSV). A-D show cells grownfor 6 days in the absence of drugs (A), or in the presence of 75 μMforskolin (B), 13 μM Segoline B (C) or 0.21 μM Eilatin (D). E and F showthe appearance of the Segoline B- and Eilatin-treated cells, 7 daysafter removal of the toxins.

As illustrated in FIG. 8 at A, this tumor cell line displays alterationsin cell growth and morphology that is characteristic of many oncogenic,transformed cell lines. The morphology of a 6 day old culture isdisorganized, the cells are small, have rounded or spindle-like shapesindicated a decreased adhesion to the substratum, and can pile up due toa loss of contact inhibition. Another characteristic feature of thetransformed phenotype, the disruption of cytoskeletal organization thatgoverns cell morphology, is illustrated in FIG. 9 (at A-B). FIG. 9 (at Aand B) shows a pair of fluorescence micrographs of fixed NIL8-HSV cellsstained with rhodamine-phalloidin to show the effects of Segoline B onF-actin organization. A: F-actin staining in control cells. B: F-actinstaining in a cell treated for 4 days with 13 μM Segoline B. As seen inthis figure, the fluorescent-staining patterns of F-actin (labeled withrhodamine-phalloidin) in 4 days old untreated NIL8-HSV cells are diffuseand lack the normal system of microfilament bundles (stress fibers) seeSpector et al., "Cell Motility and the Cytoskeleton", 13, 127-144(1989). A single application of forskolin (50-100 μM) to NIL8-HSVcultures can transiently reverse the effects of transformation, so thatthe cells acquire normal growth and morphology, and assemble stressfibers (data not shown). However, as shown in FIG. 8 at B, with time inculture the cells reverted to the transformed phenotype. In contrast,one application of 13 μM Segoline B or 0.21 μM Eilatin was sufficient topermanently reverse the effects of transformation. As illustrated inFIGS. 8 at C and D, and in FIG. 9 at B the Eudistoma alkaloids haddramatic effects on cell shape and actin organization. The treated cellsbecame large and flat and showed the typical network of stress fiberslike their normal counterparts see Spector et al., "Cell Motility andthe Cytoskeleton", 13 127-144 (1989) Furthermore, the cells maintainedtheir reduced growth and flattened morphology even seven (7) days afterdrug removal (FIG. 8 at E, and F). As illustrated in FIG. 10 at B-D,similar effects on NIL8-HSV cells were obtained with4-methylpyrido[2,3,4-kl]acridine, pyrido[2,3,4-kl]acridine and with alower concentration of Eilatin (0.08 μM) which did not exert any effecton the normal NIL8 cells.

Example VI

Biochemical studies

The results of the cell biological studies show that the six (6) newalkaloids isolated from the Red Sea tunicate Eudistoma sp. and the twosynthetic pyridoacridines represent a new class of powerful growthregulatory compounds that cause growth inhibition, differentiation andreverse transformation in cancer cell lines. The results further suggestthat these compounds act on the cAMP signaling system. To obtain moredirect evidence for this hypothesis, time inventors chose to assess theeffects of the Eudistoma alkaloids on three well-characterized metabolicsystems regulated by cAMP. These include:

1. Activity of pyruvate kinase--a key glycolytic enzyme whose activityis inhibited by cAMP-dependent phosphorylation.

2. Induction of mRNA for P-enolpyruvate carboxykinase (PEPCK)--a keyenzyme in the gluconeogenesis pathway in liver and kidney cells whosegene transcription rate is stimulated by cAMP see Hod et al., N.Y. Acad.Sci., 478, 31-45 (1986) and Hod et an., J. Biol. Chem., 263, 7747-7752(1988).

3. Growth hormone release from anterior pituitary cells which isstimulated by a number of hormonal factors that activate the adenylylcyclase system and elevate cellular cAMP levels, see, Bilezikjian etal., Endocrinology, 113, 1726-1731 (1983); Ray et al., Mol. Cell.Endocrinology, 45, 175-182 (1986) and Gabriel et al.,Neuroendocrinology, 50 170-176 (1989).

The results obtained by the inventors are summarized in FIGS. 11-13.FIG. 11 is a graph showing the effects of the Eudistoma alkaloids onpyruvate kinase activity in cultured rat hepatoma FAO cells. Enzymeactivity v/Vmax was assayed in partially purified extracts from cellsincubated for 1 h in the absence (squares) or presence of 100 μM8-(4-chlorophenylthio) cyclic AMP (CPT-cAMP)-CPT-cAMP (circles) and 26μM Segoline A (triangles) as a function of substrate concentrations(phosphoenolpyruvate, PEP). The dashed line represents the K_(m) whichmeasures the affinity of an enzyme to a substrate, defined as thesubstrate concentration that yields the half maximal reaction rate withthe enzyme, for the substrate and it was 0.14 for the control, 0.52 fortime cAMP analog, and 0.44 for Segoline A. K_(m) 's for Norsegoline andDebromoshermilamine were 0.4 and 0.46, respectively.

FIG. 12 is a graph showing the effects of Eudistoma alkaloids on levelsof PEPCK mRNA in FTO-2B rat hepatoma cells. The cells were exposed tomedium which contained: 5 μM 8-CPT-cAMP (line 4), 5, 26 and 52 μMSegoline A (lines 5-7), 2.6, 12.8 and 38.4 μM Debromoshermilamine (lines8-10) or to control medium (line 11). The level of mRNA was determinedby Northern blot analysis according to Hod et al., J. Biol. Chem., 259,15603-15608 (1984) using the plasmid pPCK10rc as a hybridization probe,and quantified by densitometric scanning of the autoradiograms which isshown on the left.

FIG. 13 is a graph showing the effects of debromoshermilamine on thesecretion of growth hormone (GH) from dispersed rat anterior pituitarycells. Cells grown in 24-well multiwell plates were incubated for 3 h infresh medium (DME) in the absence (control) or presence of 1 mM DbcAMP,and increasing concentrations of Debromoshermilamine as indicated.Hormone secretion was measured using rat GH radioimmunoassay. Each barrepresents the mean ±s.e.m of four wells.

Taken together the results presented in FIGS. 11-13 show that theEudistoma alkaloids mimic the effects of cAMP analogs in all threesystems. Segoline A, Debromoshermilamine and norsegoline were found toinhibit pyruvate kinase activity at submaximal substrate concentrations,causing a ca. 3-fold rise in the K_(m) which measures the affinity of anenzyme to a substrate, defined as the substrate concentration thatyields the half maximal reaction rate with the enzyme, for substrate(phosphoenolpyruvate) (FIG. 11). Segoline A and Debromoshermilamine werefound to stimulate the induction of PEPCK mRNA in a dose-dependentmanner (FIG. 12). Finally, Debromoshermilamine was found to stimulategrowth hormone release, again, in a dose-dependent manner (FIG. 13).Since in all cases the effects were similar to those induced by cAMPanalogs, those results support the contention that the Eudistomaalkaloids constitute a new class of potent drugs that cause growthinhibition differentiation, and reverse transformation in cancer celllines by acting on the cAMP signalling system. However, the alkaloidsdid not stimulate cAMP synthesis and did not directly interfere with theactivity of the free catalytic subunit of the cAMP-dependent proteinkinase or with the activation of the type I protein kinase holoenzyme.These results together with the striking differences between theEudistoma alkaloids and the cAMP analogs and cAMP elevating agents onlong-term cellular process, namely cell growth and differentiation,strongly suggest that the Eudistoma alkaloids affect target proteinsthat are associated with the cAMP signaling system in a novel and uniquefashion.

Biomimetic Synthesis of Pyrido[k,l]Acridines

This Example describes a biomimetic synthesis of variouspyrido[k,l]acridines, a synthesis which is likely to have a naturalequivalent, and is expected to be suitable for the preparation ofnatural and other pyrido[k,l]acridines.

Norsegoline 35 and eilatin 40 are of special interest to the inventorsbecause of their bio-activity, namely, regulation of cellular growth andan affect on cAMP mediated processes as described in Examples V and VI.

The inventors have determined that the most likely biomimetic synthesisof norsegoline 35 and other pyrido[k,l]acridines precursors 36, wouldemploy the natural o-benzoquinone 41 (or the readily, in situ,oxidiseable hydroquinone) and the tryptophan matabolite kynuramine(3-amino-4orthoaminoylend-1-proyanane) as starting materials for thesynthesis that is illustrated in FIG. 14(B). In FIG. 14(B) R₁₁ mayinclude a substituent selected from the group of phenyl, halogen,hydroxy, CO₂ -Methyl, HO-Methyl, COCH₃, CH₃, H, NHOCH₃, NO₂, NH₂ and N₃.

A. Synthesis of Benzo-Pyrido[k,l]Acridine 46

As shown in FIG. 15, in order to avoid the competition problem betweenthe two amino groups of 42 the inventors started this set of Examplesusing the symmetric 2,2'-diaminobenzophenone 45, as described byPartridge et al., J. Chem. Soc. (C), 632 (1962). Reacting catechol 44with 45 under mild oxidative conditions (aq. EtOH, NaIO₃ ; e.g. asdescribed by Tindale et al., Aust. J. Chem., 37, 611 (1948) andreferences cited therein, and by Schafeir et al., Chem. (Inter. Ed.),10, 405 (1971), likewise other oxidants may suitably be used in thesynthesis, such as CeCl₃ or Fe(SO₄)_(2;) this afforded adduct 46, aquinolino[k,l]acridine) possessing an iminoquinone functionality.Specifically, compound 45 and catechol 44 were combined (2:1 ratio) inaq. EtOH and stirred at room temperature for 24 hours in the presence ofNaIO₃ (5 equivalents). The ppt was filtered and flashed through a silicagel column to afford compound 46 (15% yield). Compound 46 was analyzedand has the following physical characteristics:

Yellow crystals (CH₂ Cl₂), mp 196°-198°, C₁₉ H₁₀ N₂ O m/z 282 (20%),νmax 1643, 1588, 1397, 1250, 980, 823 cm⁻¹. δ_(H) (CDCl₃, 500 MHz):9.13d (J=8.2) & 9.11d (j=8.2) (H-1,13), 8.76d (J=8.2) & 8.38d (J=8.2)(H-4,10), 7.97m (5H), 7.05d (J=10.5, H-7). δ_(c) 183.9s, 149.6s, 147.9s(x2), 145.3s, 144.1d, 135.6s, 133.8d, 1.33.4d, 131.8d, 131.1d (x2),130.3d, 129.3d, 127.5d, 127.0d, 124.4s, 121.1s, 111.1s.

Compound 46 was accompanied by 1:2 catechol-amine adducts, as describedin Example VIII E.

B. Synthesis of Compound 48, a Potential Precursor of Benzo-Norsegoline.

Compound 45 was reacted with 3,4-dihydroxybenzoic acid 47, as shown inFIG. 16, under the same conditions described in Example VII A to give inlow yields (5-10%) of compound a potential precursor ofbenzo-norsegoline. Compound 48 was analyzed and has the followingphysical characteristics:

A yellow gum, C₂₀ H₁₀ N₂ O₃, m/z: M-CO, 298 (47%), 298-CO₂, 254 (86%).νmax 3340, 1660, 1568, 1356, 1217 cm⁻¹, δ_(H) (CDCl₃ -d₄ -MeOH 9:1)9.15d (J=8.2) & 9.02d (J=8.2) (H-1, 13), 8.69d (J=8.2) & 8.43d (J=8.2)(H-4, 10), 7.96m (4H), 7.81t (J=8.2, 1H).

Compound 48 was accompanied by 1:2 catechol-amine adducts, vide infra.

C. Synthesis of 6-Oxo-Pyrido[k,l]acridine 49

As illustrated in FIG. 17, kynuramine 42 was reacted with catechol underthe same oxidative conditions described in Example VII A to afford apyrido[k,l]acridine compound 49 (10% yield), most likely the 6-oxo(rather than 4-oxo) derivative, with accompanying 1:2,catechol-kynuramine, adducts as described in Example VII E. Compound 49has the following physical characteristics:

A yellow gum, C₁₅ H₈ N₂ O, m/z: M+2H, 234 (100%), M, 232 (22%); νmax1647, 1621, 1578, 1340, 1272 cm⁻¹. δ_(H) (CDCl₃): 9.23d (J=5.6 Hz, H-2),8.57m (2H, 11 & 1), 8.26d (J=8 Hz, H-8), 7.93t (J=8 Hz, H-9(10)), 7.86d(J=10.6 Hz, H-4(5)), 7.79t (J=8 Hz, H-10(9)), 7.04d (J=10.5 Hz, H-5(4));δ_(c) (CDCl₃): 177.2s (CO), 151.6s, 150.2d, 147.3s, 142.3s, 142.5d,138.0s, 134.1d, 131.8d, 131.7d, 130.2s, 129.8d, 123.0d, 121.8s, 119.5d,117.4s.

D. Synthesis of Dihydropyrido[k,l]acridine Compound 51, andPyrido[k,l]acridine Compound 52

Instead of catechol 44, 3,5-cyclohexanedione 50 (formally"dihydro-1,3-dihydroxybenzene") was reacted with kynuramine 42, asillustrated in FIG. 18, to obtain in high yield (ca. 90%)dihydropyrido[k,l]acridine 51 which by oxidation with DDQ(dichlorodicyano-p-quinone) afforded the pyrido[k,l]acridine 52, aspreviously described by the inventors; see Gellerman et al., Tet.Letters, 33, 5577 (1992). Specifically, kynuramine 42 was reflaxed with1,3-cyclohexanedione 50 in the presence of an oxidant, sodiumm-nitrophenylsulfonate, in acetic acid and traces of HCl for 2 hourswhich afforded after work up compound 51 (90% yield). Compound 51 wasanalyzed and found to have the following physical characteristics:

A yellow powder, C₁₅ H₁₂ N₂, m/z M, 220 (100%); δ_(H) (CDCl₃): 8.79d(J=5.7, H-2), 8.48d (J=8, H-11), 8.20d (J=5.7, H-1), 8.11d (J=8, H-8),7.82t (J=8, H-9 or 10), 7.66t (J=8, H-10 or 9), 3.37m (4H), 2.35quin(J=6, 2H); δ_(c) 162.1s, 160.9s, 147.9d, 145.0s, 137.9s, 130.8d, 129.4d,126o6d, 122.8d, 121.9s, 118.2s, 113.6d, 34.4t, 33.8t, 22.3t.

This reaction is similar to the one reported by Partridge et al. (1962),supra, between dimedone and compound 45 except for the need for anoxidant in case of compound 51, required for the aromatization, whichmight be the driving force of the reaction.

The scope of the reactions described in Examples VIIA-VIID are expectedto be broad, namely, o-quinones, p-quinones, 2,5-dihydroxy-p-quinones,substituted derivatives of the latter compounds, as well as thecorresponding hydroquinones may be used for the preparation of naturalpyrido[k,l]acridines and their analogues.

E. Synthesis of 1:2 Adducts, Compounds 54 and 55, as PotentialPrecursors of Eilatin 40 and its Derivatives.

As mentioned in Examples VIIA and E, in some of the reactions theinventors could also identify 1:2 catechol-amine adducts. Thus, asillustrated in FIG. 19, the reaction of catechol 44 ando-aminoacetophenone 53 under the reaction conditions described inExample VIIA resulted in a 60% yield of a 1:2 adduct, compound 54.Compound 54 is asymmetric regarding the two o-amino acetophenones,namely, one molecule formed an acridine-1,2-dione system while the otherstayed as an appendix of the latter heterocycle. Compound 54 wasanalyzed and found to have the following physical characteristics:

Orange crystals mp. 206°-208° C. (CH₂ Cl₂) C₂₂ H₁₆ N₂ O, m/z: 356 (25%),νmax 1686, 1668, 1612, 1529, 1261, 786 cm⁻¹. δ_(H) (CDCl₃): 8.38d (J=8,H-8), 8.29d (J=8, H-5), 7.98d (J=8, H-3'), 7.84t (J=8, H-7), 7.77d (J=8,H-6'), 7.71t (J=8, H-6), 7.60t (J=8, H-5'), 7.27(J=8, H-4'), 6.80s(H-3), 3.19s (Me-9), 2.73s (COMe); δ_(c) (CDCl₃) 201.1s, 182.7s, 178.6s,152.2s, 150.9s, 146.5s, 146.4s, 137.9d, 133.6d, 132.6d, 131.7d, 130.6d,128.6s, 127.5s, 125.3d, 124.4d, 122.Od, 119.6s, 114.9s, 102.7d, 28.2q,15.6q.

Reacting compound 54 with BF₃ etherate provided, in high yields (70%),the symmetric pentacyclic dibenzo-1,10-phenantroline-5,6-dione 55.Functionalization of the two methyls of 55 is expected to lead toapplicable intermediates for eilatin 40 and its derivatives.Specifically, compound 54 in CH₂ Cl₂ was refluxed in the presence of BF₃Et₂ O for 24 hours to afford compound 55 after work up. Compound 55 wasanalyzed and found to have the following physical characteristics:

An amorphous yellow powder, C₂₂ H₁₄ N₂ O, m/z M+H, 339 (1%), M-2H, 336(9%), M+H-CO, 311, (94%) M-CO, 310 (84%); νmax 1686, 1500, 1422, 1308,1287, 896 cm⁻¹ ; δ_(H) (CDCl₃) 8.52d (J=8.2, H-1, 10), 8.30d (J=8.2,H-4,7), 7.92t (J=8.2, 2H), 7.70t (J=8.2, 2H), 3.19s (2Me's); δ_(c)186.2s, 153.0s, 151.2s, 149.7s, 132.6d, 132.1d, 129.6d, 129.4s, 125.5d,124.1s, 16.2q.

The 4,5-diamination of 1,2-quinones is in agreement with the reaction ofo-benzoquinone with aniline, as described by Tindale et al. (1989)supra, and Schafeir et al. (1971) supra. However, those publicationsutilized biogenic amines like 2-phenylethylamine, rather than theβ,β'-diaminoketones 42 and 45 used by the inventors, thus only yieldingpolymeric tars.

The biogenic chemistry developed in this example points to kynuramine 42and suitable quinones or hydroquinones as potential precursors foreilatin 40, norsegoline 35 and other pyrido[k,l]acridine alkaloids.

EXAMPLE VIII

Two Step Biomimetic Total Synthesis of Eilatin

The inventors have tried many approaches in attempting to synthesize thehighly bio-active molecule eilatin 40, and previously have failed.

A. Synthesis of Compounds 58, 59

Thus, as illustrated in FIG. 20, the inventors have prepared from1,10-phenanthroline-5,6-dione 56 a series of double Schiff bases 58, 59.Specifically, 1,10-phenanthroline-5,6-dione 56 was refluxed withp-anisidine 57 in acetic acid for 30 minutes. After evaporation thecrude product 58 was purified on a silica gel column (65% yield);Compound 58 was analyzed and found to have the following physicalcharacteristics:

Dark blue oil, C₂₆ H₂₀ N₄ O₂, m/z M⁺ 420 (100%), 308, (75%); λmax (CH₃CN) 605 (13800), 430 (11100), 290 (39400); IR (KBr) 1590, 1260, 1000,850, 810 cm⁻¹ ; δ_(H) (CDCl₃ -d₄ MeOH; 10:1): δ_(H) 9.61 (1H, dd, J=1.5,3.5 Hz), 9.25 (1H, dd, J=1.5, 7.5 Hz), 9.14 (1H, dd, J=1.5, 3.5 Hz),8.32 (1H, d, J=8 Hz), 8.01 (2H, dd, J=3.5, 7.5 Hz), 7.86 (1H, dd, J=1.5,7.5 Hz), 7.80 (2H, d, J=9 Hz), 7.75 (1H, d, J=8 Hz), 7.46 (2H, dd,J=3.5, 7.5 Hz), 7.45 (2H, d, J=9 Hz), 7.31 (2H, d, J=9 Hz), 7.02 (2H, d,J=9 Hz), 3.76 (3H, s), 3.59 (3H, s); δ_(c) (5% CD₃ OD/CDCl₃); 161.6s,158.6s, 155.3s, 152.3d, 151.7d, 149.Os, 144.8s, 144.1s, 139s, 135.3d,133.4d, 133.Od, 131.Os, 129.5s, 128.Od, 126.2s, 125.1d, 124.4d, 122.2d,119.5s, 117.0d, 114.5d, 56.6q, 55.9q.

The inventors have tried without success to perform a double cyclisationof these molecules 58, 59 to obtain Eilatin 40.

Observation of the NMR spectra of the compound 58, specifically thespectrum of 59, a di p-methoxyphenyl deep blue compound (compare withcompound 26 synthesized in Example III and illustrated in FIG. 2F)clearly pointed to a complex mixture of unsymmetrical conformers whichcould not easily be equilibrated by warming up 59 in solution.

B. Synthesis of Phenyl-Acididemin 64

In another approach illustrated in FIG. 21 the inventors performed adouble Skraup reaction between 4-nitro-o-phenylenediamine 60 and twomolecules of 3'-chloropropiophenone 61 to afford in ca. 15% yield4,7-diphenyl-5-nitro-1,10-phenanthroline 62. Specifically,4-Nitro-o-phenylenediamine 60 was reacted with β-chloropropiophenone 61under the Skraup reaction conditions to afford in 15% compound 62, whichwas analyzed and found to have the following physical characteristics:

An amorphous yellow powder; m/z 377 (100%) (C₂₄ H₁₅ N₃ O₂); δ_(H)(CDCl₃) δ9.32 (1H, d, J=4.7 Hz), 9.26 (1H, d, J=4.7), 8.31 (1H, s), 7.70(1H, d, J=4.7), 7.68 (1H, d, J=4.7), 7.36-7.59 (1OH, m); δ_(c) (CDCl₃):152.2d, 150.7d, 150.18, 147.68, 147.Os, 146.78, 138.28, 136.1s, 129.5d,129.3d, 129.Od, 128.6d, 127.88, 127.4d, 126.2d, 124.3s, 123.8d, 122.8d,118.88; IR(KBr): 1610, 1530, 1490, 1410, 1380, 1350, 1260, 1180, 1090,1020, 900, 850, 820, 800, 780, 760, 690, 650 cm⁻¹ ; UV (MeOH), 266, 222nm.

Heating the solution of compound 62 in dodecane at 180° under N₂ for 2hours, in the presence of (EtO)₃ P gave via the nitrene, as reported byCadogen, Quart. Rev., 22, 122 (1968), the unexpected ketone 64 which bycareful 2D NMR study (COSY, d-NOE, HMQC and HMBC) was determined to bephenyl-ascididemin. See, Kobayashi et al., Tet. Letters, 29, 1177(1988), and Moody et al., Tet., 48, 3589 (1992). The analysis ofcompound 69 disclosed the following physical characteristics:

m/z: MH⁺ 360 (100%) (C₂₄ H₁₃ N₃ O); δ_(H) (d₆ -DMSO): δ9.25 (d, H-2,J=5.0 Hz), 9.05 (d, H-5, J=5.0 Hz), 9.03 (dd, H-13, J=7.5, 1.0 Hz), 8.94(d, H-1, J=5.0 Hz), 8.55 (d, H-6, J=5.0 Hz), 8.35 (d, H-10, J=8.0 Hz),8.06 (dt, H-11, J=8.0, 1.0 Hz), 8.02 (dt, H-12, J=7.5, 1.0 Hz), 7.94bs(5H) (H-2'-6'); δ_(c) 182.1s (C8), 153.6s (C3b), 153.4s (C8a), 152.1d(C5), 150.1s (C3a), 149.8d (C2), 140.2s (C13b), 137.4d (C10), 132.3d(C11), 132Os (C7), 130.7d (C12), 128.5d (C6), 147.88 (C9a), 145.48(C7a), 128.1-128.5 (C1'-6'), 124.5d (C13), 123.38 (C13a), 117.8d (C1),117.7s (C3c); IR (KBr) 1682, 1600, 1428, 1201, 1170, 770 cm⁻¹ ; UV(MeOH); 366, 308, 264, 244, 219 nm.

Repeating the reaction under oxygen free argone gave the originallyexpected compound 63, an unstable oil, which was analyzed and found tohave the following physical characteristics: δ_(H) (CDCl₃): 8.89d(J=5.2), 8.73d (J=5.2) (H-2 & 5), 7.76d (J=8, H-13(10)), 7.50m (Ph),7.27m (2H), 6.80m, (3H), 6.68s (H-8).

Under oxygen atmosphere compound 63 was transformed into compound 64.All attempts to prepare eilatin 40 from compound 63 (e.g., by reactingit under different conditions with HN3) failed.

C. Conceptional Basis for the Synthesis of Eilatin 40

From the above data and several other experiments the inventorsconcluded that it will be difficult if not impossible to synthesizeeilatin 40, if the last step of the synthesis scheme requirescyclization of one or two aromatic rings (as in case of compounds 58 and64, respectively) because of the severe repulsion between the protonsand/or other substituents on the latter rings (both in compounds 64 and58 the appendix phenyl(s) seem to be out of plane with the rest of themolecule). Therefore the preferred synthesis should involve closure of apiperidine ring(s) which then will readily be oxidized to the requiredpyridine ring(s).

Based on biomimetic synthesis of pyrido[k,l]acridines described inExample VII, the inventors conceived of the strategy illustrated in FIG.22 for synthesizing eilatin 40, in which two kynamurine molecules or itsderivatives are reacted with O-benzoquonine, hydroquinone or theirderivatives under mild oxidative reaction conditions.

D. Two Step Biomamatic Total Synthesis of Eilatin

In carrying out the synthesis scheme for eilatin 40, described in VIIIC, above, the mono protected trifluoroacetyl kynuramine 65 was reactedwith catechol 44 under oxidative conditions (aq. EtOH, NaIO₃), asdescribed by Tindale et al., Aust. J. Chem., 37, 611 (1984) andreferences cited therein. This reaction afforded compound 66, whosestructure was determined by 2D NMR measurements (500 MHz, COSY, HMQC,HMBC) to be a 1,2-acridinedione derivative. Specifically, in thisreaction 3'-Trifluorokynuramine 65 (δ_(H) 7.62d (J=8, H-3), 7.24t &6.58t (J=8, H-4,5), 6.61d (J=8, H-6), 3.76m (2H-3'), 3.19t (2H-2') wasreacted with catechol 44 in aq. EtOH in the presence of 5 equivalents ofNaIO₃, as described by Tindale et al., supra. After 24 hours the ppt wasfiltered and chromatographed on a silica gel column to afford compound66 (ca. 15%), which was determined to have the following physicalcharacteristics: mp 249°-251° C.(CH₂ C₂), orange crystals, C₂₈ H₂₀ F₆ N₄O₅ m/z: 606, M(1.5%), 493, M-NH₂ COCF₃ (35%), 380, M-2NH₂ COCF₃ (50%);νmax 3320, 1717, 1611, 1576, 1526 cm⁻¹ ; δ_(H) CDCl₃ -d₄ -MeOH 9:1):8.52d (J=8.0, H-5), 8.30d (J=8.0, H-8), 7.97d (J=8.0, H-3'), 7.92t(J=8.0, H-7), 7.80t (J=8.0, H-6), 7.69d (J=8.0, H-6'), 7.62t (J=8.0,H-5'), 7.32t (J=8.0, H-4'), 6.67s (H-3), 3.83t, 3.67t, 3.58t, 3.33t (2Heach); δ.sub. c 201.6s (C7'), 183.1s (C1), 162.1 (COCF₃), 151.7s (C9),151.4s (C4), 147.3s (C8a), 146.2s (C4a), 138.2s (C1'), 134.3d (C5'),133.5d (C7), 131.4d (C3'), 130.8d (C8), 130.0d (C6), 128.5 (C8b), 127.5s (C2'), 125.8d (C5), 125.2d (C4'), 123.4d (C6'), 103.3d (C3), 39.3t,38.8t, 34.9t, 27.8t.

Basic treatment of compound 66 (NH₃ -MeOH, cat. DMAP) directly yieldedeilatin 40. Having in compound 66 all of the functionalities in theright position seems to have provided a strong and crucial driving forcetowards the formation of eilatin 40.

Alternatively, the last step towards the goal compound eilatin 40, couldhave been divided into two steps, namely, BF₃ etherate cyclisation tothe dibenzo-1,10-phenanthroline-5,6-dione derivative 67, as described inExample VII. Specifically, compound 67 was obtained from 66 after 24hours in CH₂ Cl₂ in the presence of BF₃ etherate. Compound 67 wasanalyzed and determined to have the following physical characteristics:an amorphous yellow powder, C₂₈ H₁₈ F₆ N₄ O₄ m/z M, 588 (1.5%); δ_(H)(CDCl₃): 8.58d (J=8, H-1 & 10), 8.46d (J=8, H-4 & 7), 8.04t (J=8) &7.82t (J=8) (H-2, 3, 8 & 9), 3.89m (2×2H), 3.78m (2×2H).

Sequentially, thereafter mild NH₃ -MeOH treatment of 67 produced eilatin40.

The readily and, very simple, biomimetic reaction described in thisexample, although only suggestive, points to kynuramine ando-benzoquinone, or hydroquinone, both natural products, as to thepotential biosynthetic precursors of eilatin 40.

Thus, while there lave been described what are the presentlycontemplated preferred embodiments of the present invention, furtherchanges and modifications could be made by those skilled in the artwithout departing from the scope of the invention, and it iscontemplated to claim all such changes and modifications.

We claim:
 1. A method for regulating cell growth, comprising:contactinga cell with an effective concentration of a compound for regulating thegrowth of the cell, said compound consisting essentially ofDebromoshermilamine.
 2. A method according to claim 1, wherein saidcompound further is combined with a biologically acceptable carrier. 3.A method according to claim 1, wherein said compound is further combinedwith a biologically acceptable carrier and said effective concentrationrange of said compound is 0.01 μM to 100 μM.
 4. A method according toclaim 3, wherein said effective concentration range is 6-32 μM.
 5. Amethod as recited in claim 1, wherein said cell is a tumor cell, andwhereby said method causes the growth of the tumor cell to suppressed.6. A method as recited in claim 2, wherein said cell is a tumor cell,and whereby said method causes the growth of the tumor cell to besuppressed.
 7. A method as recited in claim 3, wherein said cell is atumor cell, and whereby said method causes the growth of the tumor cellto be suppressed.
 8. A method as recited in claim 4, wherein said cellis a tumor cell, and whereby said method causes the growth of the tumorcell to be suppressed.
 9. A method as recited in claim 5, whereby saidmethod also induces differentiation of the tumor cell.
 10. A method asrecited in claim 6, whereby said method also induces differentiation ofthe tumor cell.
 11. A method as recited in claim 7, whereby said methodalso induces differentiation of the tumor cell.
 12. A method as recitedin claim 8, whereby said method also induces differentiation of thetumor cell.
 13. A method as recited in claim 5, whereby said method alsoinduces reverse transformation of the tumor cell.
 14. A method asrecited in claim 6, whereby said method also induces reversetransformation of the tumor cell.
 15. A method as recited in claim 7,whereby said method also induces reverse transformation of the tumorcell.
 16. A method as recited in claim 8, whereby said method alsoinduces reverse transformation of the tumor cell.
 17. A method asrecited in claim 1, wherein said cell is a transformed cell, and wherebysaid method induces reverse transformation of said transformed cell. 18.A method as recited in claim 2, wherein said cell is a transformed cell,and whereby said method induces reverse transformation of saidtransformed cell.
 19. A method as recited in claim 3, wherein said cellis a transformed cell, and whereby said method induces reversetransformation of said transformed cell.
 20. A method as recited inclaim 4, wherein said cell is a transformed cell, and whereby saidmethod induces reverse transformation of said transformed cell.
 21. Amethod as recited in claim 1, wherein said contacting comprisescontacting a plurality of cells, and whereby said method inducesinhibition of proliferation of said cells.
 22. A method as recited inclaim 2, wherein said contacting comprises contacting a plurality ofcells, and whereby said method induces inhibition of proliferation ofsaid cells.
 23. A method as recited in claim 3, wherein said contactingcomprises contacting a plurality of cells, and whereby said methodinduces inhibition of proliferation of said cells.
 24. A method asrecited in claim 4, wherein said contacting comprises contacting aplurality of cells, and whereby said method induces inhibition ofproliferation of said cells.
 25. A method for regulating cell growth,comprising:contacting a cell with an effective concentration of acompound for regulating the growth of the cell, said compound consistingessentially of Debromoshermilamine.
 26. A method according to claim 25,wherein said compound further is combined with a biologically acceptablecarrier.
 27. A method according to claim 25, wherein said compound isfurther combined with a biologically acceptable carrier and saideffective concentration range of said compound is 0.01 μM to 100 μM. 28.A method according to claim 27, wherein said effective concentrationrange is 6-32 μM.